Induc ion o Apop osis and Cell Cycle A es in Human Colon
Ca cinoma Cells by Co ema album Lea es
An onio J. León-Gonzáleza, Ma ga e M. Mansonb, Miguel López-Láza oa, Inmaculada Na a oc and
Ca men Ma ín-Co de oa,*
aDepa men o Pha macology, School o Pha macy, Uni e si y o Se ille, Spain
bDepa men o Cance S udies and Molecula Medicine, Biocen e, Uni e si y o Leices e , UK
cDepa men o Physical Chemis y, Facul y o Pha macy, Uni e si y o Se ille, Spain
[email p o ec ed]
Recei ed: Sep embe 6 h, 2013; Accep ed: Sep embe 17 h, 2013
The lea es o Co ema album (E icaceae), an endemic sh ub which g ows in A lan ic coas al a eas o he Ibe ian Peninsula, a e ich in la onoids and o he
seconda y me aboli es. Silica gel column ch oma og aphy o he e hyl ace a e ex ac om d ied lea es was pe o med and a la onic ac i e ac ion was
ob ained. The cy o oxic ac i i y o his ac ion was assessed using he colon cance cell lines HCT116 and HT29. A e 48 hou s o ea men , cell iabili y
was de e mined wi h luminescence-based ATPLi e assay, showing IC50 alues o 7.2 ± 0.7 and 6.8 ± 1.2 µg/mL, espec i ely. The s udy by low cy ome y
e ealed ha he cy o oxici y o his ac ion was media ed, a leas in pa , by induc ion o apop osis and G2/M cell cycle a es . The ac i e ac ion was hen
subjec ed o Sephadex LH-20 ch oma og aphy and wo la onoids we e sepa a ed and iden i ied as he la anone pinocemb in and 2’,4’-dihyd oxychalcone
a e UV, MS and NMR analysis.
Keywo ds: Co ema album, E icaceae, Cy o oxici y, HCT116 colon ca cinoma cell line, HT29 colon ca cinoma cell line, 2’,4’-Dihyd oxychalcone,
Pinocemb in.
Colo ec al cance is one o he leading causes o dea h wo ldwide,
and he mos common cance in he Eu opean Union, aking in o
accoun incidences in bo h sexes [1]. Due o he na ow he apeu ic
ange o 5- luo u acil (5-FU), i ino ecan and pla inum-based d ugs,
cu en ly used o colo ec al cance , esea ch in o new d ugs o
al e na i e ea men s is g owing, wi h a special in e es in na u al
p oduc s as sou ces o new an icance lead molecules.
Co ema album (L.) D. Don (E icaceae), is an endemic sh ub na i e
o he A lan ic coas o he Ibe ian Peninsula, g owing in sandy
coas al a eas om La Co uña o Cadiz. Thei acidic, as y ui s a e
sphe ical, whi e be ies which a e consumed as appe ize s, juices o
used in olk medicine. We ha e ecen ly epo ed a high con en o
phenolic compounds, mainly phenolic acids, and in i o
an ioxidan p ope ies o C. album be ies [2,3], and we ha e
ecen ly isola ed cy o oxic dihyd ochalcones om he E OAc
ex ac o he lea es [4]. The e o e, con inuing he s udy o his
plan , we ha e looked o o he cy o oxic compounds om he
lea es o his species and hei mechanism o ac ion on HCT116
and HT29 colon cance cell lines.
Subjec ion o column ch oma og aphy o he E OAc ex ac
ob ained om C. album lea es ga e a la onic ac ion (F3), which
p esen ed cy o oxic ac i i y agains HCT116 and HT29 human
colon cance cell lines. T ea men wi h F3 signi ican ly educed
cellula iabili y in a ime and dose-dependen manne (p < 0.05).
The IC50 alues a e 48 h o ea men , de e mined using ATPLi e®
luminescence-based assay we e 7.2 ± 0.7 µg/mL in HCT116 and
6.8 ± 1.2 µg/mL in HT29.
Annexin V-FITC/PI s aining and low cy ome y we e used o
de e mine he e ec s o F3 on he induc ion o apop osis in HCT116
and HT29 colon cance cells (Figu e 1). The pe cen age o HCT116
cells in apop osis was inc eased in a dose-dependen way a e 48 h
o F3 ea men , bu i was no signi ican ly a ec ed a e 24 h
Figu e 1: P oapop o ic e ec on HCT116 and HT29 cell lines a e 24 h and 48 h
ea men s wi h 0, 6 and 12 µg/mL o he ac i e ac ion (F3) o C. album lea es.
Pe cen age o cells in apop osis was analyzed by low cy ome y using he Annexin
V-FITC assay. Compa ed wi h DMSO con ol: * p < 0.05, ** p < 0.01.
(Figu e 1A). O he wise, all es ed concen a ions aised he
pe cen age o HT29 cells in apop osis, eaching a i e- old inc ease
a highe doses (Figu e 1B).
To de e mine whe he a cell cycle modi ica ion was p oduced by F3,
ea ed cells we e s ained wi h p opidium iodide and analyzed by
low cy ome y. As shown in Figu es 2A and 2B, a 12 µg/mL F3
ea men o ei he 24 h o 48 h p oduced a signi ican G2/M a es
on HCT116 cells accompanied by a educ ion in he p opo ion in S
phase. The same e ec was obse ed in HT29 cells a e 24 o 48 h
o incuba ion wi h 6 µg/mL F3 (Figu es 2C and 2D). Howe e ,
inc easing concen a ions p oduced a dec ease o his e ec on
HT29. The ac i e ac ion was hen subjec ed o Sephadex LH-20
ch oma og aphy using CH2Cl2/MeOH (1:1) o elu ion and wo
la onoids we e sepa a ed and iden i ied as he la anone,
pinocemb in, and 2’,4’-dihyd oxychalcone by compa ing hei UV,
1H NMR, and 13C NMR spec oscopic and MS da a, wi h ha in he
li e a u e [5,6].
In i o s udies ha e demons a ed ha chalcones p esen in some
medicinal plan s (e.g., isoliqui i igenin, om liquo ice) inhibi he
p oli e a ion o cance cells by inducing apop osis and blocking cell
cycle p og ession [7,8].
NPC Na u al P oduc Communica ions 2014
Vol. 9
No. 1
55 - 56
56 Na u al P oduc Communica ions Vol. 9 (1) 2014 León-González, e al.
Figu e 2: Cell cycle dis ibu ion o HCT116 and HT29 cell lines a e 24 h and 48 h
ea men s wi h 0, 6 and 12 µg/mL o he ac i e ac ion (F3) o C. album lea es.
Pe cen age o cells in G0/G1, S and G2/M phases was analyzed a e p opidium iodide
s aining by low cy ome y. Compa ed wi h DMSO con ol: * p < 0.05.
In conclusion, a c ys allized ac ion o he E OAc ex ac om
Co ema album yielded pinocemb in and 2’,4’-dihyd oxychalcone.
These isola es showed a signi ican e ec on he iabili y o wo
di e en colon cance cell lines in a low mic omola concen a ion
ange. The obse ed cy o oxici y may be due o an induc ion o
apop osis and an accumula ion o cells on G2/M cell cycle phases.
Expe imen al
Gene al: 1D and 2D NMR, B uke AVANCE-500; EI and FAB
MS, Mic omass AUTOSPECQ; UV, Shimadzu UV-1800 UV– is;
TLC, Me ck silica gel 60 F254. E OAc and n-hexane we e
pu chased om Pan eac.
Plan ma e ial: Ae ial pa s o Co ema album we e collec ed in
Sep embe 2008 in Huel a (SW Spain, 37° 04´ 10.15" N - 6° 41´
15.45" W) and iden i ied by D Díaz Ba adas. A ouche specimen
was deposi ed a he he ba ium unde no. SEV277798.
Ex ac ion and isola ion: D ied and unc ushed lea es (250 g) we e
ex ac ed in an ul asound ba h wi h E OAc (500 mL) a oom
empe a u e o 45 min. A e il a ion and e apo a ion o he
sol en unde educed p essu e, pa o his c ude ex ac (3 g) was
ac iona ed by CC o e silica gel, using n-hexane wi h a E OAc
g adien o inc easing pola i y ( om 99:1 o 90:10), o a o d 20
ac ions o 100 mL each. F ac ion 3 (F3, 200 mg) was ob ained as
yellow needles and selec ed o cell cy o oxici y assays. Ac i e
ac ion F3 was subjec ed o Sephadex LH-20 CC, using
CH2Cl2/MeOH (1:1) o elu ion. Two compounds we e isola ed and
iden i ied as pinocemb in (12 mg) and 2’,4’-dihyd oxychalcone (30
mg), on he basis o hei UV, EIMS and NMR da a.
Cell cul u e: HCT116 (p53 w adenoca cinoma) and HT29
(p53 mu an adenoca cinoma) colon ca cinoma cell lines we e
main ained a 37°C (5% CO2) in DMEM wi h 10%, / , e al
bo ine se um (In i ogen). A 20 mg/mL F3 s ock solu ion was
p epa ed in DMSO. Cells we e ea ed wi h 0-12 μg/mL F3 o
imes up o 48 h. DMSO did no exceed 0.1% in any ea men .
Cell iabili y: Cell iabili y was assessed ia measu emen o
cellula ATP le els using ATPLi e® ki (Pe kin Elme ) acco ding o
he manu ac u e ’s ins uc ions. B ie ly, HCT116 and HT29 cells
we e seeded in whi e Packa d Viewpla es-96 (1 x 104 cells pe well)
and incuba ed o e nigh o allow cell a achmen , a e which he
cells we e ea ed wi h 0-12 μg/mL F3 o 48 h. Luminescence was
di ec ly p opo ional o cell iabili y exp essed as old change om
DMSO con ol.
Flow cy ome y: Fo low cy ome y assays, 1 x 105 o 2.5 x 105
cells pe well we e seeded on 6-wells pla es, le o adhe e o e nigh
and hen ea ed wi h app op ia e concen a ions o F3 o 48 h.
Apop osis was de e mined by using he Annexin V-FITC ki
(Bende Medsys ems), as desc ibed p e iously [9]. The pe cen age
o li e, apop o ic o nec o ic cells in he whole popula ion was
de e mined using a FACScan low cy ome e (Bec on Dickinson).
O he wise, he e ec o his ac ion on he cell cycle was s udied
by analysis o DNA con en using FACScan, acco ding o a
p e iously desc ibed me hod [10].
S a is ical analysis: All samples we e assayed in 3 independen
expe imen s and esul s a e epo ed as he mean ± SEM. S a is ical
compa isons wi h DMSO ea ed cells we e made by means o
S uden ’s - es ; p alues < 0.05 we e ega ded as signi ican .
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