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 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 MO DAU Ch6m ch6m co ten khoa hoc la Nephelium lappaceum L., thuOc ho Sapindaceae Loai cay an qua thich hop a vOng nhiat dOi, la giong cay duoc Tau tien O DOng Nam A 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 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 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 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 cac san phem c6 gia tri gia tang cao (mg dung nhiau linh vut khac Geraniin duOc tach tit v6 ch6m chOrn vOi hiau suat 3,8% Ngoai tac dung chong cao huyOt ap va trdu &Ong, Geraniin c6 tac CM1C - SO 9/2016 NGHIEN ciru - UNG DUNG dung digu tri ung thU De xac dinh nang digu tri ung thU di can, bai bao 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 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 ung thu da &roc bigt tei tit nhi6u nam digu tri ung thu vu v5 hoat tinh cheng to b5o ung thU di cti LU-1 dieu tri ung thu ph6i [3,4] THVC NGHIEM 2.1 Nguyen lieu va thiel bi - Geraniin tinh sach >95% dit6c san xuat tai Ph6ng thi nghiem digrn tong nghe loc, hba du - Be KIT xac dinh VEGF (VEGF ELISA KIT) hang BioVision (Milpitas, CA, M9) cung cap - Cac d6ng to bao ung thu: 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 mOi trutng nuoi cay DMEM vei phan kern theo g6m mM L-glutamin, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), va 1,0 mM natri pyruvat, ngoai IA sung 10% fetal bovine serum - FBS (Invitrogen) va nuoi 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 DMSO (Dimethyl sulfoxide) 10% cling dutc dila vao cac giang cua khay 96 Ong a cac n6ng dO khac 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 m6i truing nuoi cay 2.2.1.2 Phuong phap xac dinh VEGF 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 60 phut - Rita bin Ian bang PBS (phosphate buffer saline) 0,01M, them 100 ill AvidinBiotin-Peroxidase Complex vac) cac gigng thi nghiem U bin a 37°C 30 phut - Rita bin 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 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 Ian da dam bio tinh chinh xac DMSO 10% luon dutc sit dung nhu' d6i cht-ing am Chat thit nao c6 Si < se ducc xem la co kha nang itc cha to bao ung thu' san sinh ygu to tao mach va gay di can VEGF 2.2.2 Xdc dinh hog tinh thong to bao ung thu'di cu'LU-1 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 m6i trutng nuoi cay DMEM (Dulbecco's Modified Eagle's Medium) %id phan kern theo g6m mM L-glutamin, 10 mM HEPES, va 1,0 mM natri pyruvat, ngoai b6 sung 10% - FBS (GIBCO) - Te bao duoc cay chuygn sau 3-5 yeti ti la (1:3) va nuoi 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 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 dia giang vdi nong t6 bao la x 105 to bao/giang - Sau 24h, 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 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 to am CO2 24h - Hinh bao vdi dO phong dai 20X dudc chup qua kinh hign vi soi ngu'oc sau Oh; 6h va 24h k6 tii 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, kh6i u co kich thudc tren 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 co the [5] Vi vay sit tao mach c6 nghia quan 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 mach manh, d6ng thoi cac tin hieu ta bao qua trung gian VEGF gop phan vao cac khia canh quan cUa khoi u nhu to bao g6c ung thu va sit khdi phat ung thu [7] Nhang dac digrn khian VEGF la muc Wet.' quan 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 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 0.8 0.6 0.4 0.2 DC - KT-03 25 KT-03 50 100 ug/m1 200 ug/mI ug/ml ug/ml Hinh 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 cac nong d0 thit mau thap hon gia tri 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 cac vet xudc (vat thuting), to bao d cac canh se có xu Wong di chuyen den chO tr6ng cho dein de lam lanh vet xutc do chiau rOng cOa vet xu'dc se clan thu hep lai Chiau phac tap Qua trinh 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 hoat chit có kh5 sit di cu cOa t6 bao BAng va hinh 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 so sanh vdi d6i chang Bang 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 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 Anh twang cOa mau Geraniin dgn su' di cif dia t6 bao ung thu LU-1 (dO phong dai 20X) Trong thi nghrern Geraniin cho thay kha nang Crc ch6 scr dich chuygn ciJa t6 bao ung tilt! ph6i LU-1 a cac n6ng khac Sau 6h mau, chieu rang vet xudc oging doi chang gi5m manh chi 54,74% so vOi thdi digm ban d6u Tuy nhien cf Ong mau Geraniin Wong di) 50 lag/ml, chi6u rOng vat xudc hau nhu'khong thu hep so voi luc Tau vdi p