cance s
A icle
Hap olid Inhibi s Tumo G ow h o Hepa ocellula
Ca cinoma h ough Rb/E2F and Ak /mTOR Inhibi ion
Jun Xing 1,2 , Vikas Bhu ia 1, Khac Cuong Bui 1, Mai Ly Thi Nguyen 1, Zexi Hu 3,
Chih-Jen Hsieh 1, Ka h in Wi s ein 4, Ma c S adle 4, Ludwig Wilkens 5, Jun Li 6,7,
Ma kus Kalesse 6,7, P zemyslaw Bozko 1,* and Ruben R. Plen z 1,8,*
1Depa men o In e nal Medicine I, Medical Uni e si y Hospi al, Ebe ha d Ka ls Uni e si ä Tübingen,
72076 Tubingen, Ge many; [email p o ec ed] (J.X.); [email p o ec ed] (V.B.);
[email p o ec ed] (K.C.B.); d [email p o ec ed] (M.L.T.N.);
[email p o ec ed] (C.-J.H.)
2The Fi s A ilia ed Hospi al o USTC, Di ision o Li e Sciences and Medicine, Uni e si y o Science and
Technology o China, He ei 230001, China
3Depa men o In e nal Medicine VIII, Medical Uni e si y Hospi al, 72076 Tübingen, Ge many;
[email p o ec ed]
4Depa men Mic obial D ugs, Helmhol z Cen e o In ec ion Resea ch GmbH (HZI), 38124 B aunschweig,
Ge many; [email p o ec ed] (K.W.); ma [email p o ec ed] (M.S.)
5Ins i u e o Pa hology, Hanno e Regional Hospi al, 30043 Hanno e , Ge many; [email p o ec ed]
6Ins i u e o O ganic Chemis y, Leibniz Uni e si y o Hanno e , 30167 Hanno e , Ge many;
[email p o ec ed] (J.L.); [email p o ec ed].de (M.K.)
7Cen e o Biomolecula D ug Resea ch, Helmhol z Cen e o In ec ion Resea ch GmbH (HZI),
38124 B aunschweig, Ge many
8Depa men o In e nal Medicine II, Klinikum B emen No d, 28755 B emen, Ge many
*Co espondence: p zemyslaw[email p o ec ed] (P.B.);
uben.plen z@gesundhei no d.de (R.R.P.); Tel.: +49-07071/29-83137 (P.B.); +49-0421/6606-1304 (R.R.P.);
Fax: +49-07071/29-4680 (P.B.); +49-0421/6606-1720 (R.R.P.)
Recei ed: 1 Decembe 2019; Accep ed: 5 Feb ua y 2020; Published: 6 Ma ch 2020


Abs ac :
Backg ound: Hepa ocellula ca cinoma (HCC) ep esen s a majo heal h bu den wi h limi ed
cu a i e ea men op ions. The e is a subs an ial unme need o de elop inno a i e app oaches
o impac he p og ession o ad anced HCC. Hap olid is a no el na u al componen isola ed om
myxobac e ia. Hap olid has been epo ed as a po en selec i e cy o oxin agains a panel o umo
cells in ecen s udies including HCC cells. The aims o his s udy a e o e alua e he an i umo e ec
o hap olid in HCC and o unde s and i s unde lying molecula mechanisms. Me hods: The e icacy
o hap olid was e alua ed in human HCC cell lines (Huh-7, Hep3B and HepG2) and xenog a umo s
(NMRI-Foxn1
nu
mice wi h injec ion o Hep3B cells). Cy o oxic ac i i y o hap olid was de e mined
by he WST-1 and c ys al iole assay. Wound healing, answell and umo sphe e assays we e
pe o med o in es iga e mig a ion and in asion o HCC cells. Apop osis and cell-cycle dis ibu ion
we e measu ed by low cy ome y. The e ec s o hap olid on he Rb/E2F and Ak /mTOR pa hway we e
examined by immunoblo ing and immunohis ochemis y. Resul s: hap olid ea men signi ican ly
inhibi ed cell p oli e a ion, mig a ion and in asion
in i o
. The epi helial–mesenchymal ansi ion
(EMT) was impai ed by hap olid ea men and he exp ession le el o N-cadhe in, imen in and
Snail was down egula ed. Mo eo e , g ow h o HCC cells
in i o
was supp essed by inhibi ion o
G1/S ansi ion, and pa ially by induc ion o apop osis. The d ug induced down egula ion o cell
cycle egula o y p o eins cyclin A, cyclin B and CDK2 and induced up egula ion o p21 and p27.
Fu he e idence showed ha hese e ec s o hap olid we e associa ed wi h Rb/E2F down egula ion
and Ak /mTOR inhibi ion. Finally,
in i o
nude mice expe imen s demons a ed signi ican inhibi ion
o umo g ow h upon hap olid ea men . Conclusion: Ou esul s show ha hap olid inhibi s he
g ow h o HCC h ough dual inhibi ion o Rb/E2F and Ak /mTOR pa hways. The e o e, hap olid
migh be conside ed as a new and p omising candida e o he pallia i e he apy o HCC.
Cance s 2020,12, 615; doi:10.3390/cance s12030615 www.mdpi.com/jou nal/cance s
Cance s 2020,12, 615 2 o 17
Keywo ds: HCC; hap olid; Rb/E2F; Ak /mTOR; EMT; cell cycle; apop osis
1. In oduc ion
Hepa ocellula ca cinoma (HCC) is one o he mos common cance s globally and i s incidence and
mo ali y a e ha e s ill been inc easing o e he pas decades [
1
]. Mo e han hal o all HCC pa ien s
a e diagnosed a ad anced s age when cu a i e ea men s a e no applicable [
2
]. Cu en ly, pallia i e
i s -line ea men s wi h y osine kinase inhibi o s like so a enib o len an inib a e a ailable [
3
]. Da a
abou immune checkpoin inhibi o s a e also p omising, bu new ea men app oaches wi h less
side-e ec s and good esponse a e s ill needed o o e come his deadly disease [3,4].
Hap olid was isola ed om he myxobac e ium Bysso o ax c uen a in 2000. Hap olid has been
epo ed as an e ec i e cy o oxic compound agains many umo cell lines including HCC cells and
i s IC50 alues a e wi hin he nanomola ange [
5
,
6
]. In his s udy, we aimed o assess he e icacy o
hap olid in HCC and o elucida e i s unde lying molecula mechanisms o unc ion, he eby p o iding
a a ionale o es ing i as a no el an icance d ug in clinical ials. This s udy is he i s epo o he
applica ion o hap olid in an in i o and in i o model o HCC.
Many p ima y pa hways al e ed in human HCC ha e been desc ibed. The e inoblas oma umo
supp esso (Rb) pa hway is equen ly dis up ed in HCC [
7
,
8
]. Classically, Rb unc ions as a cen al
playe in cell cycle egula ion [
9
,
10
]. Mi ogenic signals a e con eyed h ough cyclin-dependen kinases
(CDKs) which subsequen ly egula e Rb phospho yla ion. Phospho yla ed Rb disassocia es wi h E2F
ansc ip ion ac o s, a amily o cell-cycle- egula ed ansc ip ion ac o s which s imula e p oli e a ion,
and a e wa ds ini ia es he ansc ip ion o E2F esponsi e genes. Rb, p107 and p130 a e homologous
p o eins collec i ely known as he ‘Rb pocke p o ein amily’ which con ibu es o he egula ion o
E2F- esponsi e genes [
11
,
12
]. The key ole o he Rb/E2F pa hway in egula ing cell cycle p og ession
and con olling p oli e a ion o e s emendous po en ial o he de elopmen o he apeu ics.
The PI3K/Ak /mTOR signaling cascade is ano he majo signaling pa hway implica ed in HCC
ca cinogenesis and plays a cen al ole in d i ing umo cell p oli e a ion [
8
,
13
]. The PI3K/Ak /mTOR
signaling pa hway is a p o o ypal su i al pa hway which is cons i u i ely ac i a ed in a b oad ange o
cance s. This pa hway is in ol ed in many ypes o cellula p ocesses including su i al, p oli e a ion,
me as asis, me abolism and angiogenesis [
13
–
15
]. Gi en he equen hype ac i a ion o de egula ion
o Ak /mTOR pa hway in HCC [
15
–
17
], se e al small molecule inhibi o s a ge ing his pa hway ha e
been de eloped and a e cu en ly unde going p eclinical o clinical ials [8,18].
Ou da a sugges ha hap olid ea men has g ea an i umo ac i i y in HCC h ough dual
inhibi ion o Rb/E2F and Ak /mTOR pa hways; hus, i may be a new and p omising candida e o
sys emic ea men o HCC.
2. Ma e ials and Me hods
2.1. Cell Cul u e
Human HCC cell line Huh-7 was ob ained om he labo a o y o La s Zende , Uni e si y Hospi al
Tübingen, Tübingen, Ge many. HepG2 was ob ained om he labo a o y o Michael Bi ze , Uni e si y
Hospi al Tübingen, Ge many. Hep3B was pu chased om Leibniz Ins i u e DSMZ—Ge man collec ion
o mic oo ganisms and cell cul u es. Cell lines we e cul u ed a 37
◦
C unde a 5% CO
2
en i onmen
in DMEM (The mo Scien i ic, Da ms ad , Ge many) en iched wi h 10% e al bo ine se um (FBS)
(Bioch om, Be lin, Ge many) and an ibio ics o penicillin/s ep omycin (50 uni s/mL) (Lonza, Ve ie s,
Belgium).
Cance s 2020,12, 615 3 o 17
2.2. D ug P epa a ion and In Vi o T ea men
Hap olid was isola ed om he cul u e b o h o Bysso o ax c uen a, using he ch oma og aphic
p ocedu e desc ibed by S einme z e al. [
6
], and p epa ed as a 10 mg/mL s ock in dime hyl sul oxide
(DMSO) (AppliChem, Da ms ad , Ge many). The d ug was di ided in aliquo s, s o ed a 4
◦
C and
used o
in i o
and
in i o
expe imen s. Cells we e ea ed wi h DMSO as a con ol o wi h hap olid
in di e en concen a ions ( om 0.006 o 6
µ
g/mL) and we e analyzed a e 24 o 96 h o ce ain
expe imen s, namely cell p oli e a ion assay, c ys al iole s aining, mig a ion assay, in asion assay,
umo sphe oid assay, cell cycle analysis and apop osis assay. The p o ocols used in his s udy a e
desc ibed in de ail in Supplemen a y Ma e ials and Me hods.
2.3. Animals and T ea men
NMRI-Foxn1
nu
emale mice we e bough om Cha les Ri e Labo a o ies In e na ional (Sulz eld,
Ge many). Eigh mice we e ea ed in ape i oneally wi h hap olid (2 mg/kg body weigh , on days 1 o
3 each week); six mice we e ea ed wi h ehicle (DMSO). T ea men was s a ed a ound 10 weeks
o age and mice we e sac i iced a e 5 weeks o ea men . All ea men solu ions we e p epa ed
eshly on he day o deli e y. Heal h s a us o mice was moni o ed e e y day. Sizes o isible umo s
we e measu ed by calipe and o gan issues we e ha es ed. Tumo olume was calcula ed using
he ollowing o mula: Tumo olume V =((Wid h)
2×
(Leng h)/2). Tumo issues we e ixed in 4%
o malin o his ology.
2.4. His ology and Immunoblo ing
De ailed in o ma ion abou an ibodies and p o ocols can be ound in he Supplemen a y Ma e ials
and Me hods.
2.5. S a is ics
All he expe imen s we e epea ed 2–3 imes. The esul s we e analyzed using G aphPad P ism
e sion 7.03 (San Diego, CA, USA) and ImageJ 1.47 (Na ional Ins i u es o Heal h, Be hesda, MD, USA)
so wa e. The es s included S uden ’s - es (pai ed and unpai ed) and wo-way ANOVA. Di e ences
we e conside ed as s a is ically signi ican when he p- alue was <0.05 (*), <0.01 (**), <0.001 (***),
<0.0001 (****) o as no signi ican (ns).
2.6. S udy App o al
Mice used in his s udy we e main ained in he animal ca e o Uni e si y Hospi al Tübingen,
Tübingen, Ge many. All expe imen al p o ocols we e e iewed and app o ed by ins i u ional
guidelines o animal ca e o Uni e si y Hospi al Tübingen (p o ocol No. M7/17), and all s udies we e
pe o med acco ding o he me hods app o ed in he p o ocol.
3. Resul s
3.1. Hap olid T ea men Inhibi s P oli e a ion and Me as asis in Human HCC Cells
I was shown be o e ha hap olid (Figu e 1A) has an i umo e ec s agains a ious umo cells [
6
].
Howe e , da a abou HCC and hap olid a e limi ed. The e o e, we de e mined he cy o oxic e ec o
hap olid on h ee human HCC cell lines (Hep 3B, Huh-7 and HepG2) by WST-1 assay. In addi ion,
human ib oblas s, as noncance cells, we e simila ly es ed o display di e en ial cy o oxici y (Figu e S1).
The concen a ions o 50% o maximal inhibi ion o cell p oli e a ion (IC50) o Hep3B, Huh-7 and
HepG2 cells a e 96 h exposu e we e 0.1533, 0.0313 and 0.0618
µ
g/mL, espec i ely (Figu e 1B).
We con inued wi h concen a ions anging om 0.006 o 6
µ
g/mL o ou u he
in i o
expe imen s.
Nex , we pe o med cell p oli e a ion and c ys al iole assays. As depic ed in Figu e 1C,D and
Cance s 2020,12, 615 4 o 17
Figu e S1, Hap olid ea men supp essed cell p oli e a ion o Hep3B, Huh-7 and HepG2 cell lines in a
dose- and ime-dependen manne .
Cance s 2020, 12, x FOR PEER REVIEW 4 o 16
S1, Hap olid ea men supp essed cell p oli e a ion o Hep3B, Huh-7 and HepG2 cell lines in a dose-
and ime-dependen manne .
Figu e 1. Hap olid ea men inhibi s p oli e a ion o human hepa ocellula ca cinoma (HCC) cells.
(A) Chemical s uc u e o hap olid. (B) Cy o oxic e ec o hap olid (IC50): Hep3B, Huh-7 and HepG2
cells we e ea ed o 96 h wi h inc easing concen a ions o hap olid (0.001 o 18 µg/mL). WST-1 assay
was pe o med o analyze cellula iabili y, DMSO was used as nega i e con ol. (C) Cell p oli e a ion
o HCC cells ea ed wi h he indica ed concen a ions o hap olid o DMSO measu ed by WST-1
assay. The ela i e cell numbe was no malized wi h he con ol. (D) HCC cells we e ixed and s ained
wi h c ys al iole 48 and 96 h a e hap olid ea men . Da a ep esen means ± SEM o a leas h ee
independen expe imen s. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figu e 1.
Hap olid ea men inhibi s p oli e a ion o human hepa ocellula ca cinoma (HCC) cells. (
A
)
Chemical s uc u e o hap olid. (
B
) Cy o oxic e ec o hap olid (IC50): Hep3B, Huh-7 and HepG2 cells
we e ea ed o 96 h wi h inc easing concen a ions o hap olid (0.001 o 18
µ
g/mL). WST-1 assay was
pe o med o analyze cellula iabili y, DMSO was used as nega i e con ol. (
C
) Cell p oli e a ion o
HCC cells ea ed wi h he indica ed concen a ions o hap olid o DMSO measu ed by WST-1 assay.
The ela i e cell numbe was no malized wi h he con ol. (
D
) HCC cells we e ixed and s ained wi h
c ys al iole 48 and 96 h a e hap olid ea men . Da a ep esen means
±
SEM o a leas h ee
independen expe imen s. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fu he mo e, wound healing and Ma igel answell chambe assays we e conduc ed o examine
he e ec o hap olid on he cell me as asis. In all cell lines, hap olid ea men induced signi ican
inhibi ion o wound closu e (Figu e 2A). Hap olid signi ican ly educed he numbe o in asi e cells,
as illus a ed in Figu e 2B. Thus, ea men wi h hap olid can e ec i ely supp ess he mig a ion and
in asion in human HCC cells. Fu he mo e, we explo ed an
in i o
h ee-dimensional umo sphe oid
model o mimic physiologic issue’s mic oen i onmen o in es iga ing d ug e iciency o hap olid.
As we can see in Figu e 3A, hese esul s u he con i med ha hap olid ea men signi ican ly
Cance s 2020,12, 615 5 o 17
inhibi ed he sphe oid g ow h and in asion in Hep3B and Huh-7 cells. Ou expe imen s showed ha
incuba ion o he Huh-7 cell line wi h hap olid o 10 days supp essed he g ow h o sphe oid size bu
wi h no eg ession, whe eas simila incuba ion o he Hep3B cell line eadily esul ed in educ ion
o umo sphe oid size a he concen a ion o 0.6
µ
g/mL as ea ly as 4 days. Taken oge he , hese
da a sugges ha hap olid can signi ican ly inhibi p oli e a ion, mig a ion and in asion o human
HCC cells. In gene al, Hep3B cells shows o e all s onge inhibi o y e ec unde hap olid ea men
compa ed o Huh-7 and HepG2 cells.
Cance s 2020, 12, x FOR PEER REVIEW 5 o 16
Fu he mo e, wound healing and Ma igel answell chambe assays we e conduc ed o examine
he e ec o hap olid on he cell me as asis. In all cell lines, hap olid ea men induced signi ican
inhibi ion o wound closu e (Figu e 2A). Hap olid signi ican ly educed he numbe o in asi e cells,
as illus a ed in Figu e 2B. Thus, ea men wi h hap olid can e ec i ely supp ess he mig a ion and
in asion in human HCC cells. Fu he mo e, we explo ed an in i o h ee-dimensional umo
sphe oid model o mimic physiologic issue’s mic oen i onmen o in es iga ing d ug e iciency o
hap olid. As we can see in Figu e 3A, hese esul s u he con i med ha hap olid ea men
signi ican ly inhibi ed he sphe oid g ow h and in asion in Hep3B and Huh-7 cells. Ou expe imen s
showed ha incuba ion o he Huh-7 cell line wi h hap olid o 10 days supp essed he g ow h o
sphe oid size bu wi h no eg ession, whe eas simila incuba ion o he Hep3B cell line eadily
esul ed in educ ion o umo sphe oid size a he concen a ion o 0.6 µg/mL as ea ly as 4 days.
Taken oge he , hese da a sugges ha hap olid can signi ican ly inhibi p oli e a ion, mig a ion and
in asion o human HCC cells. In gene al, Hep3B cells shows o e all s onge inhibi o y e ec unde
hap olid ea men compa ed o Huh-7 and HepG2 cells.
Figu e 2. Inhibi ion o mig a ion and in asion in hap olid- ea ed HCC cells. (A) Wound healing
assay was pe o med o Hep3B, Huh-7 and HepG2 cells a e hap olid (0.06, 0.6 and 6 µg/mL)
Figu e 2.
Inhibi ion o mig a ion and in asion in hap olid- ea ed HCC cells. (
A
) Wound healing assay
was pe o med o Hep3B, Huh-7 and HepG2 cells a e hap olid (0.06, 0.6 and 6
µ
g/mL) ea men .
The do ed lines ep esen edges o he wound. Pho og aphs we e aken a 0 and 24 h unde ligh
mic oscope (40
×
magni ica ion). The mig a ion index was calcula ed as desc ibed in Sec ion 2and
plo ed in ba g aphs. (
B
) HCC cells we e seeded in Ma igel answell in asion chambe s and
ea ed wi h indica ed concen a ions o hap olid o 48 h o in es iga e he e ec on in asi eness.
The numbe o cells ha in aded h ough he memb ane was de e mined unde ligh mic oscope
(200
×
magni ica ion). Scale ba s: 100
µ
m. In asion index was calcula ed as desc ibed in Sec ion 2
and plo ed in ba g aphs. Da a ep esen means
±
SEM o a leas h ee independen expe imen s.
*p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Cance s 2020,12, 615 6 o 17
3.2. Hap olid T ea men Impai s EMT in HCC Cells
Mo eo e , based on he indings ha hap olid ea men signi ican ly inhibi ed he HCC cell
mo ili y, we con inued o in es iga e he impac o hap olid in EMT. A he molecula le el, EMT
is equen ly de e mined by he exp ession le el o pu a i e EMT ma ke s: he up egula ion o
mesenchymal ma ke s, such as cy oskele al p o ein imen in and cell su ace p o ein N-cadhe in, and
he down egula ion o epi helial ma ke s, including he junc ion p o ein E-cadhe in [
19
,
20
]. In o de o
check whe he EMT is impai ed a e hap olid ea men , we ea ed HCC cells wi h inc easing dosages
o hap olid o 96 h and compa ed wi h nega i e con ol. In bo h Hep3B and Huh-7 cells, hap olid
ea men esul ed in dec eased exp ession o he mesenchymal p o eins N-cadhe in and imen in as
assessed by Wes e n blo (Figu e 3B). Howe e , he exp ession o epi helial p o ein E-cadhe in was
only sligh ly up egula ed (Figu e 3B). Thus, we nex checked he exp ession o Snail, a zinc- inge EMT
ansc ip ion ac o ha unc ions as a po en ep esso o E-cadhe in [
21
]. As shown in Figu e 3B, he
le el o Snail was ma kedly diminished a e hap olid ea men . These changes indica e ha hap olid
ea men pa ially impai s EMT in HCC cells.
Cance s 2020, 12, x FOR PEER REVIEW 6 o 16
ea men . The do ed lines ep esen edges o he wound. Pho og aphs we e aken a 0 and 24 h unde
ligh mic oscope (40× magni ica ion). The mig a ion index was calcula ed as desc ibed in Sec ion 2
and plo ed in ba g aphs. (B) HCC cells we e seeded in Ma igel answell in asion chambe s and
ea ed wi h indica ed concen a ions o hap olid o 48 h o in es iga e he e ec on in asi eness.
The numbe o cells ha in aded h ough he memb ane was de e mined unde ligh mic oscope
(200× magni ica ion). Scale ba s: 100 µm. In asion index was calcula ed as desc ibed in Sec ion 2 and
plo ed in ba g aphs. Da a ep esen means ± SEM o a leas h ee independen expe imen s. * p <
0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
3.2. Hap olid T ea men Impai s EMT in HCC Cells
Mo eo e , based on he indings ha hap olid ea men signi ican ly inhibi ed he HCC cell
mo ili y, we con inued o in es iga e he impac o hap olid in EMT. A he molecula le el, EMT is
equen ly de e mined by he exp ession le el o pu a i e EMT ma ke s: he up egula ion o
mesenchymal ma ke s, such as cy oskele al p o ein imen in and cell su ace p o ein N-cadhe in,
and he down egula ion o epi helial ma ke s, including he junc ion p o ein E-cadhe in [19,20]. In
o de o check whe he EMT is impai ed a e hap olid ea men , we ea ed HCC cells wi h
inc easing dosages o hap olid o 96 h and compa ed wi h nega i e con ol. In bo h Hep3B and Huh-
7 cells, hap olid ea men esul ed in dec eased exp ession o he mesenchymal p o eins N-cadhe in
and imen in as assessed by Wes e n blo (Figu e 3B). Howe e , he exp ession o epi helial p o ein
E-cadhe in was only sligh ly up egula ed (Figu e 3B). Thus, we nex checked he exp ession o Snail,
a zinc- inge EMT ansc ip ion ac o ha unc ions as a po en ep esso o E-cadhe in [21]. As
shown in Figu e 3B, he le el o Snail was ma kedly diminished a e hap olid ea men . These
changes indica e ha hap olid ea men pa ially impai s EMT in HCC cells.
Figu e 3. Con .
Figu e 3. Con .
Cance s 2020,12, 615 7 o 17
Cance s 2020, 12, x FOR PEER REVIEW 7 o 16
Figu e 3. Hap olid ea men inhibi s umo sphe oids g ow h and in asion and pa ially impai s
epi helial–mesenchymal ansi ion (EMT) in human HCC cells. (A) Th ee-dimensional umo
sphe oid assay was pe o med o e alua e e ec s o hap olid on HCC cell p oli e a i e and in asi e
abili y in ex acellula -ma ix-like en i onmen . The ep esen a i e images o sphe oids a e shown.
The changes o sphe oids size we e moni o ed and quan i ied up o 10 days and plo ed in ba g aphs.
(B) Hep3B and Huh-7 cells we e ea ed wi h DMSO o hap olid (0.06, 0.6 and 6 µg/mL) o 96 h. The
exp essions o EMT ma ke s (N-cadhe in, E-cadhe in, imen in and Snail) we e analyzed by
immunoblo ing. Wes e n blo ing bands we e quan i ied by Image J and no malized o hei
espec i e β-ac in hen compa ed wi h ehicle- ea ed con ols. Da a ep esen means ± SEM o a
leas h ee independen expe imen s. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
3.3. Hap olid Inhibi s G1/S T ansi ion and Pa ially Induces Apop osis o HCC Cells
To igu e ou he unde lying mechanisms o he an ip oli e a i e e ec o hap olid, cell cycle
and cell apop osis we e analyzed. Fluo escence-ac i a ed cell so ing (FACS) assay was ca ied ou
o de e mine he cell cycle p o iles upon exposu e o di e en concen a ions o hap olid a e 48 and
96. Figu e 4A shows he dis ibu ion o cell cycle p o iles a e hap olid ea men in all h ee HCC
cell lines. The pe cen ages o he sub-G1-, G1-, S- and G2-M popula ion a e lis ed in Table S1. In Huh-
7 and HepG2 cells, hap olid ea men esul ed in a signi ican accumula ion o cells in G1 phase
accompanied wi h a dec ease o he cell numbe s in S and G2-M phase, which indica ed a block o
G1/S p og ession (Figu e 4A). Al hough Hep3B cells also accumula ed in he G1 phase a lowe
concen a ion (0.06 µg/mL), a highe concen a ions o he compound ea men , a la ge p opo ion
o he cells accumula ed in he sub-G1 phase, sugges ing an induc ion o apop osis (Figu e 4A), which
was also e idenced by ollowing expe imen s. O e all, hap olid ea men inhibi s he G1/S ansi ion
and esul s in ma ked cell cycle a es in all es ed cell lines. Mo eo e , hap olid ea men leads o
g ow h a es , la e on inducing conside able apop osis in Hep3B cells.
To u he subs an ia e cell cycle analysis esul s, we nex in es iga ed he kine ic exp ession o
cell cycle checkpoin p o eins ia Wes e n blo (Figu e 4B). Consis en wi h he blocking o G1- o-S
phase ansi ion, we ound ha he exp ession le els o bo h cyclin A and cyclin B dynamically
dec eased.
Figu e 3.
Hap olid ea men inhibi s umo sphe oids g ow h and in asion and pa ially impai s
epi helial–mesenchymal ansi ion (EMT) in human HCC cells. (
A
) Th ee-dimensional umo sphe oid
assay was pe o med o e alua e e ec s o hap olid on HCC cell p oli e a i e and in asi e abili y in
ex acellula -ma ix-like en i onmen . The ep esen a i e images o sphe oids a e shown. The changes
o sphe oids size we e moni o ed and quan i ied up o 10 days and plo ed in ba g aphs. (
B
) Hep3B
and Huh-7 cells we e ea ed wi h DMSO o hap olid (0.06, 0.6 and 6
µ
g/mL) o 96 h. The exp essions
o EMT ma ke s (N-cadhe in, E-cadhe in, imen in and Snail) we e analyzed by immunoblo ing.
Wes e n blo ing bands we e quan i ied by Image J and no malized o hei espec i e
β
-ac in hen
compa ed wi h ehicle- ea ed con ols. Da a ep esen means
±
SEM o a leas h ee independen
expe imen s. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
3.3. Hap olid Inhibi s G1/S T ansi ion and Pa ially Induces Apop osis o HCC Cells
To igu e ou he unde lying mechanisms o he an ip oli e a i e e ec o hap olid, cell cycle
and cell apop osis we e analyzed. Fluo escence-ac i a ed cell so ing (FACS) assay was ca ied ou
o de e mine he cell cycle p o iles upon exposu e o di e en concen a ions o hap olid a e 48
and 96. Figu e 4A shows he dis ibu ion o cell cycle p o iles a e hap olid ea men in all h ee
HCC cell lines. The pe cen ages o he sub-G1-, G1-, S- and G2-M popula ion a e lis ed in Table S1.
In Huh-7 and HepG2 cells, hap olid ea men esul ed in a signi ican accumula ion o cells in G1
phase accompanied wi h a dec ease o he cell numbe s in S and G2-M phase, which indica ed a block
o G1/S p og ession (Figu e 4A). Al hough Hep3B cells also accumula ed in he G1 phase a lowe
concen a ion (0.06
µ
g/mL), a highe concen a ions o he compound ea men , a la ge p opo ion o
he cells accumula ed in he sub-G1 phase, sugges ing an induc ion o apop osis (Figu e 4A), which
was also e idenced by ollowing expe imen s. O e all, hap olid ea men inhibi s he G1/S ansi ion
and esul s in ma ked cell cycle a es in all es ed cell lines. Mo eo e , hap olid ea men leads o
g ow h a es , la e on inducing conside able apop osis in Hep3B cells.
Cance s 2020,12, 615 8 o 17
Cance s 2020, 12, x FOR PEER REVIEW 8 o 16
Figu e 4. Hap olid ea men inhibi s G1/S ansi ion and induces cell cycle a es in HCC cells. (A)
HCC cells we e ea ed wi h ehicle (DMSO) o inc easing concen a ions o hap olid o 48 and 96 h,
ixed and s ained wi h p opidium iodide (PI) and subjec ed o low cy ome ic analysis.
Rep esen a i e cell cycle his og ams a e p esen ed (le panel). Cell cycle dis ibu ion in a ious
phases is de e mined and shown ( igh panel). (B) Cyclin A and B exp ession le els we e measu ed
by Wes e n blo ing. Images ep esen a i e o a leas h ee independen expe imen s a e shown.
Addi ionally, annexin V and PI s aining was pe o med on li e cells o de e mine whe he he
cell-cycle-a es pheno ype was associa ed wi h he induc ion o apop osis. We no iced ha exposu e
o hap olid dose-dependen ly inc eased apop o ic cells in Hep3B cell line (Figu e 5A,B). Mo eo e ,
p o eoly ic clea age o poly (ADP- ibose) polyme ase (PARP) as an indica i e ma ke o apop osis
was e alua ed by quan i a i e measu emen [22]. Immunoblo ing e ealed dis inc PARP clea age
a e hap olid ea men in Hep3B cells (Figu e 5C,D). In e es ingly, Huh-7 and HepG2 cells a e no
sensi i e o he apop osis-inducing e ec s o hap olid (Figu es S2 and S3). These esul s con i m ou
p e ious s udies and sugges ha Huh-7 and HepG2 cells a e ela i ely esis an o apop osis and
mainly unde go cell cycle a es unde hap olid ea men , while hap olid induces no iceable
apop o ic cell dea h in Hep3B cells ollowing cy os a ic e ec .
Figu e 4.
Hap olid ea men inhibi s G1/S ansi ion and induces cell cycle a es in HCC cells. (
A
)
HCC cells we e ea ed wi h ehicle (DMSO) o inc easing concen a ions o hap olid o 48 and 96 h,
ixed and s ained wi h p opidium iodide (PI) and subjec ed o low cy ome ic analysis. Rep esen a i e
cell cycle his og ams a e p esen ed (le panel). Cell cycle dis ibu ion in a ious phases is de e mined
and shown ( igh panel). (
B
) Cyclin A and B exp ession le els we e measu ed by Wes e n blo ing.
Images ep esen a i e o a leas h ee independen expe imen s a e shown.
To u he subs an ia e cell cycle analysis esul s, we nex in es iga ed he kine ic exp ession o cell
cycle checkpoin p o eins ia Wes e n blo (Figu e 4B). Consis en wi h he blocking o G1- o-S phase
ansi ion, we ound ha he exp ession le els o bo h cyclin A and cyclin B dynamically dec eased.
Addi ionally, annexin V and PI s aining was pe o med on li e cells o de e mine whe he he
cell-cycle-a es pheno ype was associa ed wi h he induc ion o apop osis. We no iced ha exposu e
o hap olid dose-dependen ly inc eased apop o ic cells in Hep3B cell line (Figu e 5A,B). Mo eo e ,
p o eoly ic clea age o poly (ADP- ibose) polyme ase (PARP) as an indica i e ma ke o apop osis
was e alua ed by quan i a i e measu emen [
22
]. Immunoblo ing e ealed dis inc PARP clea age
a e hap olid ea men in Hep3B cells (Figu e 5C,D). In e es ingly, Huh-7 and HepG2 cells a e no
sensi i e o he apop osis-inducing e ec s o hap olid (Figu es S2 and S3). These esul s con i m ou
Cance s 2020,12, 615 9 o 17
p e ious s udies and sugges ha Huh-7 and HepG2 cells a e ela i ely esis an o apop osis and
mainly unde go cell cycle a es unde hap olid ea men , while hap olid induces no iceable apop o ic
cell dea h in Hep3B cells ollowing cy os a ic e ec .
Cance s 2020, 12, x FOR PEER REVIEW 9 o 16
Figu e 5. Hap olid ea men induces conside able apop osis in Hep3B cells. (A) Hep3B cells we e
ea ed wi h DMSO o hap olid (0.06, 0.6 and 6 µg/mL) o 48 and 96 h, and he le el o apop osis was
measu ed by s aining wi h annexin V and PI using low cy ome y. Rep esen a i e FACS
measu emen s a e p esen ed. (B) The apop o ic posi i e cells we e calcula ed and plo ed in ba
g aph. (C) Exp ession o PARP and clea ed PARP (a ma ke o apop osis) was measu ed by Wes e n
blo ing. (D) Kine ic analysis o PARP clea age was pe o med by Wes e n blo ing. Da a ep esen
means ± SEM o a leas h ee independen expe imen s. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <
0.0001.
3.4. Dual Inhibi ion o Rb/E2F and Ak /mTOR Pa hways by Hap olid
Based on he e ec o hap olid on cell cycle, we nex examined he exp ession le els o cell cycle
checkpoin p o eins and cell cycle modula ion ac o s by Wes e n blo analysis. Fi s , we checked he
exp ession le el o Rb, which plays a cen al ole in cell cycle egula ion, pa icula ly in ini ia ing
DNA eplica ion and di ision [9]. Figu e 6A demons a ed ha —aside om he Rb-de icien Hep3B
cell line—a e hap olid ea men he le el o p-Rb exp ession dec eased and Rb changed om he
hype phospho yla ed o m o he hypophospho yla ed o m in bo h Huh-7 and HepG2 cell lines.
Mo eo e , he p o ein le el o E2F-1, he downs eam a ge o Rb, which is he essen ial ansc ip ion
ac o ha egula es cell cycle p og ession and s imula es p oli e a ion [23], was conside ably
inhibi ed a e exposu e o hap olid in all h ee HCC cells. Inhibi ion o Rb phospho yla ion and
educ ion in le els o ee E2F-1 appea o play an impo an ole in HCC g ow h a es . Hap olid
ea men also esul ed in a no iceable down egula ion o cyclin-dependen kinase 2 (CDK2) in all
h ee HCC cells and up egula ion o cyclin-dependen kinase inhibi o s (CDKIs) p21 and p27 in bo h
Hep3B and HepG2 cells. We also ound up egula ion o p21 bu no p27 in Huh-7 cells (Figu e 6A).
Nex he phospho-Se 10-his one H3 was checked as a speci ic ma ke o mi osis. As expec ed, in all
es ed HCC cells, p-his one H3 exp ession was ema kably down egula ed (Figu e 6A). Taken
Figu e 5.
Hap olid ea men induces conside able apop osis in Hep3B cells. (
A
) Hep3B cells we e
ea ed wi h DMSO o hap olid (0.06, 0.6 and 6
µ
g/mL) o 48 and 96 h, and he le el o apop osis was
measu ed by s aining wi h annexin V and PI using low cy ome y. Rep esen a i e FACS measu emen s
a e p esen ed. (
B
) The apop o ic posi i e cells we e calcula ed and plo ed in ba g aph. (
C
) Exp ession
o PARP and clea ed PARP (a ma ke o apop osis) was measu ed by Wes e n blo ing. (
D
) Kine ic
analysis o PARP clea age was pe o med by Wes e n blo ing. Da a ep esen means
±
SEM o a leas
h ee independen expe imen s. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
3.4. Dual Inhibi ion o Rb/E2F and Ak /mTOR Pa hways by Hap olid
Based on he e ec o hap olid on cell cycle, we nex examined he exp ession le els o cell cycle
checkpoin p o eins and cell cycle modula ion ac o s by Wes e n blo analysis. Fi s , we checked he
exp ession le el o Rb, which plays a cen al ole in cell cycle egula ion, pa icula ly in ini ia ing
DNA eplica ion and di ision [
9
]. Figu e 6A demons a ed ha —aside om he Rb-de icien Hep3B
cell line—a e hap olid ea men he le el o p-Rb exp ession dec eased and Rb changed om he
hype phospho yla ed o m o he hypophospho yla ed o m in bo h Huh-7 and HepG2 cell lines.
Mo eo e , he p o ein le el o E2F-1, he downs eam a ge o Rb, which is he essen ial ansc ip ion
ac o ha egula es cell cycle p og ession and s imula es p oli e a ion [
23
], was conside ably inhibi ed
a e exposu e o hap olid in all h ee HCC cells. Inhibi ion o Rb phospho yla ion and educ ion
in le els o ee E2F-1 appea o play an impo an ole in HCC g ow h a es . Hap olid ea men
also esul ed in a no iceable down egula ion o cyclin-dependen kinase 2 (CDK2) in all h ee HCC
Cance s 2020,12, 615 16 o 17
24.
Du ne , A.; Andjelko ic, M.; Bu ge ing, B.M.; Hemmings, B.A.; Thomas, G. P o ein kinase B localiza ion and
ac i a ion di e en ially a ec S6 kinase 1 ac i i y and euka yo ic ansla ion ini ia ion ac o 4E-binding
p o ein 1 phospho yla ion. Mol. Cell. Biol. 1999,19, 4525–4534. [C ossRe ]
25.
Pe e son, R.T.; Beal, P.A.; Comb, M.J.; Sch eibe , S.L. FKBP12- apamycin-associa ed p o ein (FRAP)
au ophospho yla es a se ine 2481 unde ansla ionally ep essi e condi ions. J. Biol. Chem.
2000
,
275, 7416–7423. [C ossRe ]
26.
Kodama, T.; Newbe g, J.Y.; Kodama, M.; Rangel, R.; Yoshiha a, K.; Tien, J.C.; Pa sons, P.H.; Wu, H.;
Finegold, M.J.; Copeland, N.G.; e al. T ansposon mu agenesis iden i ies genes and cellula p ocesses d i ing
epi helial-mesenchymal ansi ion in hepa ocellula ca cinoma. P oc. Na l. Acad. Sci. USA
2016
,113,
E3384–E3393. [C ossRe ]
27.
Mi , N.; Jayachand an, A.; Dhungel, B.; Sh es ha, R.; S eel, J.C. Epi helial- o-Mesenchymal T ansi ion: A
Media o o So a enib Resis ance in Ad anced Hepa ocellula Ca cinoma. Cu . Cance D ug Ta ge s
2017
,17,
698–706. [C ossRe ]
28.
E an, G.I.; Vousden, K.H. P oli e a ion, cell cycle and apop osis in cance . Na u e
2001
,411, 342–348.
[C ossRe ]
29.
Hochegge , H.; Takeda, S.; Hun , T. Cyclin-dependen kinases and cell-cycle ansi ions: Does one i all? Na .
Re . Mol. Cell Biol. 2008,9, 910–916. [C ossRe ]
30.
Hydb ing, P.; Malumb es, M.; Sicinski, P. Non-canonical unc ions o cell cycle cyclins and cyclin-dependen
kinases. Na . Re . Mol. Cell Biol. 2016,17, 280–292. [C ossRe ]
31.
B eunig, C.; Muelle , B.J.; Umansky, L.; Wahl, K.; Ho mann, K.; Lehne , F.; Manns, M.P.; Ban el, H.; Falk, C.S.
BRa and MEK inhibi o s di e en ially egula e cell a e and mic oen i onmen in human hepa ocellula
ca cinoma. Clin. Cance Res. 2014,20, 2410–2423. [C ossRe ] [PubMed]
32.
Liu, C.Y.; Hsieh, C.H.; Kim, S.H.; Wang, J.P.; Ni, Y.L.; Su, C.L.; Yao, C.F.; Fang, K. An indolylquinoline
de i a i e ac i a es DNA damage esponse and apop osis in human hepa ocellula ca cinoma cells. In . J.
Oncol. 2016,49, 2431–2441. [C ossRe ]
33.
He ze , K.; Ho mann, T.G.; Teu el, A.; Schimanski, C.C.; Moehle , M.; Kanzle , S.; Schulze-Be gkamen, H.;
Galle, P.R. IFN-alpha-induced apop osis in hepa ocellula ca cinoma in ol es p omyelocy ic leukemia
p o ein and TRAIL independen ly o p53. Cance Res. 2009,69, 855–862. [C ossRe ] [PubMed]
34.
Calzone, L.; Gelay, A.; Zino ye , A.; Rad anyi, F.; Ba illo , E. A comp ehensi e modula map o molecula
in e ac ions in RB/E2F pa hway. Mol. Sys . Biol. 2008,4, 173. [C ossRe ]
35.
Ha bou , J.W.; Dean, D.C. The Rb/E2F pa hway: Expanding oles and eme ging pa adigms. Genes De .
2000
,
14, 2393–2409. [C ossRe ] [PubMed]
36.
E angelou, K.; Ha aki, S.; Ko sinas, A. E2F ansc ip ion ac o s and diges i e sys em malignancies: How
much do we know? Wo ld J. Gas oen e ol. 2014,20, 10212–10216. [C ossRe ] [PubMed]
37.
Ri adenei a, D.B.; Mayhew, C.N.; Thanga el, C.; So illo, E.; Reed, C.A.; G aña, X.; Knudsen, E.S. P oli e a i e
supp ession by CDK4/6 inhibi ion: Complex unc ion o he e inoblas oma pa hway in li e issue and
hepa oma cells. Gas oen e ology 2010,138, 1920–1930. [C ossRe ]
38.
Hsu, I.C.; Tokiwa, T.; Benne , W.; Me cal , R.A.; Welsh, J.A.; Sun, T.; Ha is, C.C. p53 gene mu a ion and
in eg a ed hepa i is B i al DNA sequences in human li e cance cell lines. Ca cinogenesis
1993
,14, 987–992.
[C ossRe ]
39.
Li, W.; Sanki, A.; Ka im, R.Z.; Thompson, J.F.; Soon Lee, C.; Zhuang, L.; McCa hy, S.W.; Scolye , R.A. The ole
o cell cycle egula o y p o eins in he pa hogenesis o melanoma. Pa hology 2006,38, 287–301. [C ossRe ]
40.
Abukhdei , A.M.; Pa k, B.H. P21 and p27: Roles in ca cinogenesis and d ug esis ance. Expe Re . Mol. Med.
2008,10, e19. [C ossRe ]
41.
Choi, A.R.; Kim, J.H.; Yoon, S. Co igendum o "Sensi iza ion o Cance Cells h ough Reduc ion o To al Ak
and Down egula ion o Salinomycin-Induced pAk , pGSk3 be a, pTSC2, and p4EBP1 by Co ea men wi h
MK-2206". BioMed Res. In . 2015,2015, 138260. [C ossRe ] [PubMed]
42. Mann, K.K.; Colombo, M.; Mille , W.H., J . A senic ioxide dec eases AKT p o ein in a caspase-dependen
manne . Mol. Cance The . 2008,7, 1680–1687. [C ossRe ] [PubMed]
43.
Adachi, M.; Ka sumu a, K.R.; Fujii, K.; Kobayashi, S.; Aoki, H.; Ma suzaki, M. P o easome-dependen
dec ease in Ak by g ow h ac o s in ascula smoo h muscle cells. FEBS Le . 2003,554, 77–80. [C ossRe ]

Cance s 2020,12, 615 17 o 17
44.
Mendoza, M.C.; E , E.E.; Blenis, J. The Ras-ERK and PI3K-mTOR pa hways: C oss- alk and compensa ion.
T ends Biochem. Sci. 2011,36, 320–328. [C ossRe ] [PubMed]
45.
Gedaly, R.; Angulo, P.; Chen, C.; C easy, K.T.; Spea , B.T.; Hundley, J.; Daily, M.F.; Shah, M.; E e s, B.M.
The ole o PI3K/mTOR inhibi ion in combina ion wi h so a enib in hepa ocellula ca cinoma ea men .
An icance Res. 2012,32, 2531–2536. [PubMed]
©
2020 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access
a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion
(CC BY) license (h p://c ea i ecommons.o g/licenses/by/4.0/).