Nghiên cứu khả năng ức chế tế bào ung thư lu 1 (ung thư phổi) và MCF 7 (ung thư vú) của hoạt chất geraniin từ vỏ quả chôm chôm việt nam (nephelium lappaceum l )

1111
1=r1
11111MirW
)

NGHIEN
KHA NANG trc CHE TE BAO UNG THU' LU-1
(UNG THU' PHOI) VA MCF
, 7?(UNG THU VU)
Hog
CHAT GERANIIN TOP VO QUA CHOM CHOM VIII NAM
(Nephelium lappaceum L.)
ANTIMETASTA EFFECTS ON HUMAN LUNG(LU-1) AND BREAST (MCF-7)
CANCER CELL LINES OF GERANIIN FROM RAMBUTAN PEEL
(NEPHELIUM LAPPACEUM L.)
Hoang Than Hoai Thu', Vu Thi Thu Hal, Trgn Thi Thanh Thtlyl,
Iran Van Hie-U1, Nguyin Thi Nga2, Do Thi Thio2
'Ph6ng Thi nghiam trong diem Cong ngha Loc, hoa dau
'Man C6ng ngha sinh hoc - Vian Han lam Khoa hoc va cOng nghe Viet Nam
DO'n t6a soan 19/08/2016

ABSTRACT
Current chemotherapeutic drugs kill cancer cells mainly by inducincellular necrosis. However,
they become ineffective once cancer cell has the ability to metastasize, hence the poor prognosis and high mortality rate. Therefore, the purpose of this study was to evaluate the antimetastatic potential of Geraniin which was extracted from rambutan fruit on lung and breast carcinoma
cells. Several antimetastasis assays were carried out and Geraniin was shown to effectively
reduce the migration of LU-1 cells and to inhibit MCF-7 cells secreting VEGF (Vascular
Endothelial Growth Factor). The presence of polyphenol compounds in the plant is critically
important in the inhibition of the migration of cancer cells, along with the involvement of apoptosis induction. Hence, Geraniin could be a valuable candidate in the treatment of metastatic cancers.
TO' khoa: geraniin, ung tint ph6i, ung

vu, qua chom chom.

1. MO DAU
Ch6m ch6m co ten khoa hoc la Nephelium
lappaceum L., thuOc ho Sapindaceae. Loai
cay an qua nay thich hop trong a vOng nhiat
dOi, la giong cay duoc trong Tau tien O DOng
Nam A. 6 Viet Nam, chOm ch6m cita Mat
Nam tap trung chit ye'u a cac tinh phia Nam
vOi san luting khoang 358.000 tan Iran tong
dian tich gan 21.146,1ha trong do mot s6 tinh
nhu {Jong Nai khoang
san xuat
200.000 tan, Ban Tre 64.493 tan, Vinh Long
16.053 tan, Tien Giang 3.603 tan [1]. Thi
truting chOm chOm phan Ian fiat thy trong
ntidc, 30% san luting chOm chOm hang nam

38

du'oc xuat kh'eu chi, yeti sang Trung Qu6c,
Singapore va mot so nudc chau Au, M9. Khoi
luting v6 chOm chOm udc tinh bang khoang
45% - 50% [2] so voi kh6i luting qua, vi the vOi
san luting 6 cac tinh phia Nam khoang
358.000 tan thi khoi luting v6 chOrn chom bi
!JO di udc firth khoang 161.000 - 179.000 tan/
nam. Day la ngu6n nguyen lieu tiem nang de
c6 the xif 19 v6 Oa thai thanh cac san phem
c6 gia tri gia tang cao (mg dung trong nhiau
linh vut khac nhau.

Geraniin duOc tach tit v6 ch6m chOrn vOi
hiau suat 3,8%. Ngoai tac dung chong cao
huyOt ap va trdu &Ong, Geraniin con c6 tac

CM1C - SO 9/2016


NGHIEN ciru - UNG DUNG
dung trong digu tri ung thU. De xac dinh khi
nang trong digu tri ung thU di can, bai bao nay
nghien cau !chi nang itc chg t'e` bao ung thu
MCF7 sin sinh ygu to tang true ng not mo
mach mau la ygu to c6 khi nang kich thich
qua trinh tang sinh mach mau. Vai tro cua
VEGF (Vascular Endothelial Growth Factor)
dei voi hien tu'ong tan tao mach trong ung thu
da &roc bigt tei tit nhi6u nam nay trong digu
tri ung thu vu v5 hoat tinh cheng to b5o ung
thU di cti LU-1 trong dieu tri ung thu ph6i [3,4].
2. THVC NGHIEM
2.1. Nguyen lieu va thiel bi
- Geraniin do tinh sach >95% dit6c san
xuat tai Ph6ng thi nghiem trong digrn tong
nghe loc, hba du.
- Be KIT xac dinh VEGF (VEGF ELISA
KIT) do hang BioVision (Milpitas, CA, M9)
cung cap.
- Cac d6ng to bao ung thu: do GS. TS. J.
M. Pezzuto, Trutng Dai hoc Hawaii v5 Giao
su Jeanette Maier, truOng Dai hoc Milan, Italia

cung cap.
- Glutamin, natri pyruvat, fetal bovine
serum (Invitrogen, Carlsbad, CA, M9).
2.2. Phu'Ong phap nghien cOu
2.2.1. Nghian cat, kha nang tit the to bac)
ung thu'MCF7 san sinh yeu to VEGF
2.2.1.1. Phuong phap Man nuoi A xu li mau
tren to bao MCF7
- Tg bao MCF7 du'qc nuoi Gay trong mOi
trutng nuoi cay DMEM vei thanh phan kern
theo g6m 2 mM L-glutamin, 10 mM HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), va 1,0 mM natri pyruvat, ngoai ra
IA sung 10% fetal bovine serum - FBS
(Invitrogen) va nuoi trong to am CO2 a digu
kien 37°C, 5% CO2. Them vac) cac gigng thi
nghiem luting to bao phu h6p (trong 180 ill
m6i biking). De qua dem cho to bao moc bam
day IA de the phu khoang 80%.
- 10µl H202 dugc dila vac) tat ca cac gigng
thi nghiem. Deng thoi, chat this (10 til) pha
trong DMSO (Dimethyl sulfoxide) 10% cling
dutc dila vao cac giang cua khay 96 Ong a
cac n6ng dO khac nhau. Met khay 96 gigng
khac kh6ng c6 chat the,' nhu'ng c6 to bao (180
ul), 10 µl H202 va 10 ul DMSO 10% se dutc

CNI1C -

so 9/2016

sit dung lam dei chang am.
nuoi to bao da
Sau 24 h xit li mau, dich
&roc thit chat du6c thu nhan de xac dinh sit
co mat cua VEGF trong m6i truing nuoi cay.
2.2.1.2. Phuong phap xac dinh VEGF trong
dich n& nuoi ca'y tg bao
atqc thuc hien dila tren VEGF ELISA Kit
(BioVision) theo dOng Wong dAn cua nha san
xuat.
- Them 100 µl mau thit (mei twang nuoi
cay) vac) cac giang thi nghiem dia 96 Ong
(thuOc be kit). U ban a 37°C tong 90 phut.
- Loai b6 dich not va them 100 ul khang the
khang VEGF gan biotin vao cac Ong thi
nghiem. U ban a 37°C trong 60 phut.
- Rita bin 5 Ian bang PBS (phosphate
buffer saline) 0,01M, them 100 ill AvidinBiotin-Peroxidase Complex vac) cac gigng thi
nghiem. U bin a 37°C trong 30 phut.
- Rita bin 5 Ian bAng PBS 0,01M, them 90
c6 chat TMB (hydroclorit hydrat) vao cac
gigng thi nghiem. U ban a 37°C, tranh anh
sang trong 20-25 phut.
- Dung phin (mg bAng 100 µl stop solution. Doc kat qua ten may Tecan GENios Pro
microplate reader a bit& song 450 nm.
- Kha nang itc cha bao san sinh VEGF
du6c tinh theo c6ng thac:
OD(chat thit)
SI =
OD(d6i chang am)

- Cac phep this &roc lap lai 3 Ian da dam
bio tinh chinh xac. DMSO 10% luon dutc sit
dung nhu' d6i cht-ing am. Chat thit nao c6 Si <
1 se ducc xem la co kha nang itc cha to bao
ung thu' san sinh ygu to tao thanh mach va
gay di can VEGF.
2.2.2. Xdc dinh hog tinh thong to bao ung
thu'di cu'LU-1 trong didu tri ung thu'phol
2.2.2.1. Phuong phap nuoi cay tg bao in vitro
- Cac d6ng to bao ung thu ph6i LU-1 &roc
nuoi cay duti dang don 16p trong m6i trutng
nuoi cay DMEM (Dulbecco's Modified Eagle's
Medium) %id thanh phan kern theo g6m 2 mM
L-glutamin, 10 mM HEPES, va 1,0 mM natri
pyruvat, ngoai ra b6 sung 10% - FBS
(GIBCO).
- Te bao duoc cay chuygn sau 3-5 ngay yeti
ti la (1:3) va nuoi trong to am CO2 a di& kien
37°C, 5% CO2.
39


_LL

G DU

2.2.2.2. Phucing phap tray xudc xac Binh kha

3. ick qui va thio luau

nang chong di a/ coa to bao ung thu

3.1. Xac Binh kha nang tic the to bac ung
thuMCF7 san sinh yeU teicli can VEGF coa
mau thi nghiOm

- To bao dudc nuOi cay trong dia 6 giang
vdi nong do t6 bao la 1 x 105 to bao/giang.
- Sau 24h, khi ta bao dan d6u, tao vat xu'Oc
(vat thu'ong) tren b6 mat kip ta bao don Op
bang Tau nhon dm tip micropippet. Rica bang
PBS d6 loaf ba ta bao khong barn day.
- Du'a mau thi nghiem vao mai gigng nuoi
cay vdi cac nong do khec nhau: 50 µg/ml; 25
DMSO duoc sit dung lam
og/ml va 12,5
d6i chCrng am. T6 bao du'oc nu6i tiap trong to
am CO2 trong 24h.
- Hinh anh ta bao vdi dO phong dai 20X
dudc chup qua kinh hign vi soi ngu'oc sau Oh;
6h va 24h k6 tii khi o mau thi nghiem vdi ta
bac. Hinh anh du'oC phan tich bang phan
mem ImageJ de xac Binh kha nang thu hep
vat xikic cua t6 bao dudi tac Ong cua mau.
Kha nang Crc ch6 di cu coa mau duoc xac dinh
bang % sit giam d6 r6ng vat xu'dc a giang
mai, so vdi chi6u Ring vat xubc a thoi digm
ban d6u. Vat xubc cang thu hep thi hoat tinh
Crc cha sit di chuye'n t6 bao ung thu dia mau
nghien citu cang y6u va ngu'oc lai.

Trong benh ung thu, khi kh6i u co kich
thudc tren 2 mm, cac mach mau d6' cung
cap oxy va dinh du'ong la rat can thiat va tao
di6u kien de cac ta bao ung thu )(aril Ian tit
kh6i u dan cac co quan khac trong co the [5].
Vi vay sit tao mach c6 9 nghia quan trong
trong su phat than die kh6i u cling nhu'stf di
can [6]. Yau to tang tru'dng nOi mO mach
mau - VEGF la mot yau to tao mach va tang
tinh them thanh mach manh, d6ng thoi cac
tin hieu ta bao qua trung gian VEGF con gop
phan vao cac khia canh quan trong cUa khoi
u nhu to bao g6c ung thu va sit khdi phat
ung thu [7]. Nhang dac digrn nay khian
VEGF la muc Wet.' quan trong trong dieu tri
ung thu. Trong nghien diu cua chUng t6i,
kha nang Crc ch6 VEGF dia Geraniin du'oc
xac dinh.
Dich not cua ta bao MCF7 sau khi dtiqc
xir Ii mau se du'Oc phi] len giang ELISA cua
130 KIT xac dinh VEGF. Tian hanh thi nghiem
theo dung hu'ong d5n cua nha san xuat
chi:mg toi de thu du'oc kat qua nhu sau.

Kha Fling (cc the t6 bao MCF7 san sinh
VEGF so vdi d6i chang
1.2
0.4

7:1

1

1
0.8
0.6
0.4
0.2
0
DC -

KT-03 25 KT-03 50 100 ug/m1 200 ug/mI
ug/ml
ug/ml

Hinh 1. Kha nang (lc che'te. bao MCF7 san sinh VEGF cae Geraniin
Kat qua tren cho thay mau Geraniin (KT03) th6 hien kha nang Crc dig ta bao ung thu
MCF7 san xuat yau to di can VEGF manh
nhat a [long d6 this mau la 200 µg/m1 vdi gia

40

tri SI dat 0,824. 6 cac nong d0 thit mau thap
hon gia tri nay tang clan cho thay kha nang Crc
ch6 giam clan va dat thap nhat a nor-1g d0 25
gia tri SI la 0,936.

CNHC - SO 9/2016

HIE
Nhu vay, mAu Geraniin the hien hoat tinh
O'c ch6 di can thong qua kh5 nang Oc ch6 to
bao ung thu MCF7 shn sinh yau t6 VEGF.
3.2. Xac dinh hog tlnh chang te. bao ung
thu'di cuLU-1
Sy' di can cua ung thtf la mot qua trinh

GD
ung tht.t di can. Philo:mg phap tray xudc xac
dinh khh nang di chuyan cOa to bao.

6 cac

vet xudc (vat thuting), to bao d cac canh se
có xu Wong di chuyen den chO tr6ng cho
dein khi de lam lanh vet xutc do do chiau
rOng cOa vet xu'dc se clan thu hep lai. Chiau

phac tap. Qua trinh nay lien quan chat the

rOng cuA vet xutc khong 131 thu hep hay thu

vdi sit di cLf(migration), xam 16n va barn dinh

hep ch'em neu hoat chat co kha nang ch6ng

cOa to bao ung thu... Do do hoat chit có kh5

sit di cu cOa t6 bao. BAng 1 va hinh 2 the

nang UC ch6 sit di cu cOa t6 bao ung thu
cOng ph5n anh phan nao kh5 nang itc ch6

hien kh5 nang Cfc ch6 ski di chuyen to bao
ung thif khi so sanh vdi d6i chang.

Bang 1. Kh6 nang uc the su di chuyen to bao ung thu cua rn6u nghien citu
N6ng dd ( g/m1)

%

lic chs skid! chuyen

12,5

30.10

25

51.92

50

52.40

• 12.514/m1
II 25 talaml
■SO warn!

Hinh 2. Kha nang i1c the sit di chuygn to bao ung thu cOa mgu nghien

CNHC - So 9/2016

COU


6h

Oh

24h

N i ng 004 mau: 12.5 µg/m1

Nang (10 thil. mill: 25

NOng d tiler mau: 50 lig/m1
Hinh 3.

Anh twang cOa mau Geraniin dgn su' di cif dia t6 bao ung thu LU-1
(dO phong dai 20X)

Trong thi nghrern nay Geraniin cho thay
kha nang Crc ch6 scr dich chuygn ciJa t6 bao
ung tilt! ph6i LU-1 a cac n6ng do khac nhau.
Sau 6h 0 mau, chieu rang vet xudc oging
doi chang gi5m manh chi con 54,74% so vOi
thdi digm ban d6u. Tuy nhien cf Ong 0 mau

Geraniin Wong di) 50 lag/ml, chi6u rOng vat

xudc hau nhu'khong thu hep so voi luc Tau vdi
p <0,05. o nong do 25 fig/ml, chi6u r(ing vet
xiRic co thu hep !pi tuy nhien van dat Tan
80,52% so vdi thdi di6m Oh. Sau 24h u mau,
chi6u rang vet xuac tai n6ng do 50 va 25
µg/ml Wring cludng va cao hon rat nhieu so
vdi doi chang khOng u mau.

CNHC

- sti 9/2016


NGHIE C

Kat qua tran cho thay mAu Geraniin c6 kha
nang (ic the st/ di chuyen coa to bac) ung thu
phOi th6ng qua kha nang lam cham sit thu
hep dO rOng vet xutc trong thi nghiem. Dieu
do chUng to Geraniin c6 kha nang Uc the sit
di cu cua to bao ung thu ph6i LU-1.
4. KET LUAN
Da khao sat anh hutng ceta Geraniin d6i
voi sit di chuyen coa to bao ung thu phOi LU1 va vdi sit san xuat yeu to gay di can VEGF
cita to bao ung thu MCF7. Kat qua cho thay,
Geraniin the hien hoat tinh tot ch6ng lai nhCmg
dac tinh ten cita to bao ung thu ph6i va ung
thu vu. Day la nhCmg kat luan ban dau al the

ang dung hoat chat geraniin chit xuat tit v6
ch6m chom trong dieu tri ung thu.
Lai cam cm
Xin chan thanh cam co ban chi, nhiem
chucmg trinh H6a dutc (de tai
CNHD.DT.053/14-15) cua BO Cong thutng
da ho trd kinh phi thut hien de tai nay.

Tai liOu tham khao
1. Ta Minh Tuan va Doan Kau Tien,
Nghien cUu kha nang clap (mg nhu cau thi
trutng xuat khau xoai tuii OBSCL vao Trung
qu6c, Ket qua nghien cau KHCN Rau qua
2007 - ViOn NC CAQ mien Nam, Nha xuat
ban Nang nghiep, 2007.

CNHC - SO 9/2016

DU

2. Julio A Solis-Fuentes, Guadalupe
Camey-Ortiz, Mafia Del Rosario HernándezMedal, Francisco Perez-Mendoza, Carmen
Duran-De-Bamia, Composition, phase behavior and thermal stability of natural edible fat
from rambutan (Nephelium lappaceum L.)
seed, Bioresource Technology, 2010; 101(2) :
799-803.
3. Dong X Z, Zhang M, Wang K, Liu P,
Guo D H, Zheng X L, Ge X Y Sanguinarine
inhibits Vascular Endothelial Growth Factor
release by generation of reactive oxygen

species in MCF-7 human mammary adenocarcinoma
cells,BioMed
Research
International, 2013.
4. Tang F Y, Nguyen N, Maydani M.,
Green tea catechins inhibit VEGF induced
angiogenesis in vitro through suppression of
ve-cadherin phosphorylation and inactivation
of Akt molecule, Int.J.Cancer, 2003; 106:871878.
5. Carmeliet P., VEGF as a key mediator
of angiogenesis in cancer, Oncology 2005;
3:4-10.
6. Verheul HM, Pinedo HM, The role of
vascular endothelial growth factor (VEGF) in
tumor angiogenesis and early clinical development of VEGF-receptor kinase inhibitors.
Clin Breast Cancer, 2000;1(1):S80-4.
7. Goel HL, Mercurio AM, VEGF targets
the tumour cell, Nat Rev Cancer 2013;
13(12):871-82.