Endah Puji Septisetyani, Muthi’ Ikawati, Barinta Widaryanti and Edy Meiyanto)
Endah Puji Septisetyani, et al.
Apoptosis Mediated Cytotoxicity of Curcumin Analogues
PGV-0 and PGV-1 in WiDr Cell Line Endah Puji Septisetyani, Muthi’ I kaw ati, Barinta Widaryanti and Edy ) Meiyanto*Cancer Chemoprevention Research Center, Faculty of Pharmacy, Gadjah Mada University, * ) Yogyakarta, I ndonesia Corresponding author, e-mail:
Abst ra c t
Previous st udy has report ed t hat colon cancer can be blocked by ant i- inflam m at ory drugs t rough t he dow n regulat ion of cyclooxy- genase 2 ( COX 2) expression by which t hey capable of inducing
′-hydroxy-3′- apopt osis. Tw o analogues of curcum in, PGV- 0 ( 2,5- bis- ( 4 ′- m et hoxy) - benzilidine- cyclopent anone) and PGV- 1 ( 2,5- bis- ( 4
′,5′-dim ethyl)-benzilidine-cyclopentanone) have the better hydroxy,3 pot encies of ant i- inflam m at ory effect s t han curcum in. Bot h PGV- 0 and PGV- 1 also show t he m uch st ronger inhibit ion of COX 2 act ivit ies t han curcum in and aspirin in vivo. I n t his st udy, t he COX 2 inhibit ion and apopt osis induct ion effect s of PGV- 0 and PGV- 1 in WiDr colon cancer cell line were explor ed t o discover pot ent chem oprevent ive agent s for cancer colon t reat m ent . I nduct ion of pro- apopt ot ic prot ein Bax expression was also exam ined t o underst and t he role of anot her m echanism of apopt osis induct ion. The result showed t hat I C50 values
μM of PGV- 0 and PGV- 1 in WiDr cell det erm ined by MTT assay were 45 and 8 μM, respectively. Moreover, evidence of apoptosis exam ined by double st aining w it h et hidium brom ide- acrydine orange indicat ed t hat bot h PGV- 0 and PGV- 1 caused apopt ot ic bodies at inhibit ory concent rat ions. On t he ot her hand, im m unocyt ochem ist ry assay indicat ed t hat PGV- 0 significant ly decreased COX 2 expression, but cont radict ory PGV- 1 did not show any inhibit ion. PGV- 0 also induced higher Bax expression t han PGV- 1. These findings suggest t hat PGV- 1 is t he m ost prospect ive chem oprevent ive agent alt hough it act ed via independent inhibit ion of COX 2 expression pat hway. Therefore, furt her m echanism of growt h inhibit ion and apopt osis induct ion of PGV- 1 is im port ant t o be explored.
Ke y w or ds: PGV- 0, PGV- 1, WiDr cell line, apopt osis, Bax, Cyclooxygenase 2
I nt roduc t ion
Previous st udy has report ed t hat colon cancer can be blocked by ant i- inflam m at ory drugs t hrough t he dow n regulat ion of cyclooxygenase 2 ( COX 2) expression by which t hey capable of inducing apopt osis ( Chao et al., 2005) . Curcum in, t he yellow pigm ent
Apoptosis Mediated Cytotoxicity.............
in t urm eric, show s t he ant i- inflam m at ory effect and also exhibit s t he suppression of COX 2 expression in different cancer cells including colon cancer cells ( Aggarwal et al., 2005) . Moreover, t wo analogues of
′-hydroxy-3′-m ethoxy)-benzilidine- curcum in, PGV- 0 ( 2,5- bis- ( 4 cyclopent anone) and PGV- 1 ( 2,5- bis- ( 4 ′-hydroxy,3′,5′-dim ethyl)- benzilidine- cyclopent anone) have t he bet t er pot encies of ant i- inflam m at ory effect s t han curcum in. Bot h PGV- 0 and PGV- 1 also showed t he m uch st ronger inhibit ion of COX 2 act ivit ies t han curcum in and aspirin in vivo ( Molnas Team , 2001) . Therefore, in present st udy t he COX 2 inhibit ion effect s of PGV- 0 and PGV- 1 in WiDr colon cancer cell line were explor ed t o discover pot ent chem oprevent ive agent s for colon cancer t reat m ent .
Up regulat ion of pro- apopt ot ic prot ein i.e. Bax is anot her way t o induce apopt osis occurrence. Bax pore form at ion is im por t ant for t he release of cyt ochrom e C, one of apopt osis m ediat or. On t he ot her hand, Bax act ivit y is inhibit ed by ant i- apopt ot ic prot ein such as Bcl- 2 and Bcl- xL which could form het erodim er wit h Bax. Thus, suppression of ant i- apopt ot ic prot ein expression m ediat es t he form at ion of Bax pore and induces apopt osis ( King, 2000) .
I nhibit ion of COX 2 expression and induct ion of pro- apopt ot ic prot ein Bax could induce t he cancer cells t o undergo apopt osis. Cancer cells are able t o hinder apopt osis because of m ut at ion or change in norm al physiological propert ies. Consequent ly, cell cycle progression would be act ively occurs and uncont rollable ( Hanahan and Weinberg, 2000) . Moreover, com pounds t hat have good pot ency t o sensit ize t um or cells t o induce apopt osis are considered t o be prospect ive chem oprevent ive agent s ( Chao et al., 2005) .
M a t e ria ls a nd M e t hod Ch e m ica ls a n d Ce lls
Curcum in w as obt ained from Sigm a, PGV- 0 and PGV- 1 were obt ained from Curcum in Research Cent er ( CRC) , Facult y of Pharm acy, GMU. Dim et hylsulfoxide ( DMSO) , MTT [ 3- ( 4,5- dim et hylt hiazol- 2yl) - 2,5- diphenyl- t et razolium brom ide] , and ot her chem icals w ere purchased from Sigm a. Dulbecco’s m odified Eagle m edium ( DMEM) , RPMI 1640, fet al bovine serum ( FBS) , penicillin- st rept om ycin, t rypsin- EDTA 1x were purchased from Gibco. The colon cancer cell line, WiDr, was obt ained from Parasit ological Depart m ent of Medical Facult y and t he breast cancer cell line, T47D, was obt ained from NAI ST, Japan. COX 2 ant ibody was obt ained from DACO and Bax ant ibody was obt ained from Cell Signalling. All of reagent s for luciferase assay w ere provided by Laborat ory of Anim al Science, NAI ST, Japan.
Proceeding Molecular Targeted Therapy Symposium 2008
49 Endah Puji Septisetyani, et al.
Pla sm id
The NF- kB- responsive luciferase report er const ruct and t he NF- κB expression vector, pCMX-lacZ, was a kind gift from Prof. Kawaichi, NAI ST, Japan.
Sa m ple pr e pa r a t ion
Test sam ple solut ions of curcum in, PGV- 0, and PGV- 1 ( 50.000 m M) were prepared by dissolving each com pound in DMSO. Serial dilut ions of sam ple solut ions w it h cult ure m edium w ere prepar ed im m ediat ely before in vit ro assays were perform ed.
Ce ll via bilit y
Cell viabilit y w as det erm ined by using MTT assay. WiDr cells 3 ( 3x10 cells/ w ell) w ere dist ribut ed int o 96 well- plat es and incubat ed for 48 hours. Aft er rinsed t he cells using phosphat e buffer saline ( PBS) , cells were t reat ed wit h t he sam ples and aft er 24 hours incubat ion MTT reagent was added. St opper reagent was added aft er form azan form at ion prior t o MTT reduct ion. The absorbance of each well was m easured using ELI SA reader at 595 nm .
Apopt osis a ssa y 4 Aft er 48 hours incubat ion, WiDr cells ( 5x10 cells/ w ell) w ere
t reat ed wit h 30 m M curcum in, 45 m M PGV- 0, and 10 m M PGV- 1 for 15 hours. Aft er incubat ion, cells were st ained for 5 m inut es wit h μg/ m l acrydine orange – et hydium brom ide st aining solut ion ( each 5 in PBS) and viewed im m ediat ely by fluorescence m icroscope. Apopt ot ic cells which had lost t heir m em brane int egrit y appeared orange and showed m orphological feat ures of apopt osis. Cells were ident ified as apopt ot ic on t he basis of specific m orphological crit er ia, including condensat ion and fragm ent at ion of chrom at in, and form at ion of apopt ot ic bodies.
I m m u n ocyt och e m ist r y a ssa y 4 WiDr cells ( 5x10 cells/ w ell) w er e seeded int o coverslip in 24
w ells- plat e. Follow ing 48 hours incubat ion, cells w ere t reat ed w it h 30 m M curcum in, 45 m M PGV- 0, and 10 m M PGV- 1 for 15 hours. The cells at t ached on coverslip’s surface were been t reat ed wit h im m unocyt ochem ist ry reagent according t o t he m anufact urer’s prot ocol.
Tr a n sfe ct ion a n d lu cife r a se a ssa y 4 T47D cell were seeded at a concent rat ion of 2.5 x 10 cells/ well
in 24- well plat e. Aft er 21 hours incubat ion, refresh t he m edium wit h serum - free m edium and cont inue incubat ion for 4 hours, and t hen cells were t ransfect ed wit h 0.8 μg containing pNFκB-TAL-Luc and pCMX- lac Z and 2 μl of lypofectam ine 2000 (I nvitrogen) each well.
Apoptosis Mediated Cytotoxicity.............
Aft er 5 hours incubat ion m edium were refreshed wit h cult ure m edium cont aining 10% FBS and cont inue incubat ion. At 24 hours aft er μM PGV-1, t ransfect ion cell were t reat ed wit h m edium cont aining 1.5
μM curcum in, 15 μM PGV-0 and 1 ng/ m l of TNF-α. 24 hours after
20 t reat m ent cells were ly sat e using picagene ly sis buffer and luciferase act ivit y was m easured using lum inom et er
Re sult s Effe ct of PGV - 0 a n d PGV - 1 on ce ll gr ow t h
Cell v iabilit y assay was done t o det erm ine t he inhibit ory concent rat ion ( I C 50 ) of PGV- 0, PGV- 1, and also t he reference curcum in in WiDR cells. All of t hese com pounds showed growt h inhibit ory effect in dose dependent m anner. The I C 50 values of PGV- 0, PGV- 1, and
μM, respectively. PGV-1 exhibited the curcum in were 45, 8, and 27 st rongest grow t h inhibit ory effect am ong t he com pounds ( Fig.1) .
Figure 1. Cell viabilit y assay of ( A) Curcum in, ( B) PGV- 0, ( C) PGV- 1 in WiDr
cells. Result showed t hat curcum in, PGV- 0, and PGV- 1 had t he I C 50 μM, 45 μM, and 8 μM, respectively. values of 27Proceeding Molecular Targeted Therapy Symposium 2008
51 Endah Puji Septisetyani, et al.
Effe ct of PGV - 0 a n d PGV - 1 on a popt osis
All of curcum in, PGV- 0, and PGV- 1 were capable of inducing apopt osis at inhibit ory concent rat ion ( Fig. 2) . The green fluorescence indicat es t he viable cells while t he orange- red fluorescence indicat es t he deat h cells. Apopt ot ic cells show t he occurrence of chrom at in condensat ion and t he orange- red apopt ot ic bodies. PGV- 1 appeared t o have t he st rongest apopt ot ic effect .
Figure 2. Apopt osis induct ion of curcum in and pent agam avunons in WiDr
μM cell. ( A) cont rol cells, ( B) , ( C) , and ( D) cell t reat ed wit h 30 μM PGV-0, and 10 μM PGV-1 at incubation tim e 15 h. curcum in, 50I n h ibit ion of N F- k B a ct iva t ion
This st udy w as prelim inary st udy t o predict t he probabilit y of inhibit ion of NF- kB act ivat ion before we do t he sam e experim ent in WiDr cells. Wit h respect t o apopt osis induct ion, t he luciferase assay also showed t he sim ilar result w here PGV- 1 showed t he st rongest inhibit ion on NF- KB act ivat ion ( Fig. 3) . Curcum in showed t he int er-
Figure 3. Suppression on NF- B act ivat ion in T47D cells. ( A) cont rol cell κ induced by TNF- α, (B), (C), (D) cells treated with 20 μM curcum in, 1.5 μM PGV-1, 10 μM PGV- 0, (E) control cell without TNF- α. PGV-1 showed the strongest suppression on NF- κB act ivat ion induced by TNF- α.
Apoptosis Mediated Cytotoxicity.............
m ediat e effect , while PGV- 0 did not show any significant inhibit ion. Verificat ion of t his result should be done by t he real exam inat ion in WiDr cells.
I n du ct ion of Ba x e x pr e ssion
To underst and t he furt her m echanism of apopt osis induct ion, im m unocyt ochem ical assay was done t o det erm ine t he change of Bax expression. The result showed t hat curcum in induced Bax expression on highest level follow ed by PGV- 0. I n cont rast , PGV- 1 didn’t show significant induct ion ( Fig. 4) . PGV- 1 m ight be act t rough t he suppression of ant iapopt osis prot ein such as Bcl- 2 and Bcl- xl.
I n h ibit ion of COX 2 e x pr e ssion
I m m unocyt ochem ical analysis on COX 2 indicat ed t he high level of COX 2 expression in cont rol cells, show ed by darken brown colour. Curcum in and PGV- 0 show ed significant inhibit ion on COX 2 expression, showed by bluish color. I nt erest ingly, PGV- 1 didn’t showed sim ilar response alt hough it gave t he st rongest growt h inhibit ory effect and showed t he bet t er ant i- inflam m at ory effect t han curcum in in vivo ( Fig. 5) ( Molnas Team , 2001) .
Disc ussions
Previous st udy show ed t hat curcum in inhibit ed p65/ Rel A κB dependent transcriptional activity, and expression expression, NF- of NF- κB-dependent anti-apoptotic genes. However, curcum in also induced apopt osis in p65 over- expressed HCT116 hum an colon cancer cells. These findings indicat e t hat curcum in induces apopt osis t rough
κB independent m echanism (Collet and Cam pbell, 2006). We NF- hypot hesized t hat t he curcum in analogue PGV- 0 induces apopt osis t rough independent NF- κB / Rel A transcriptional activity since PGV-0 did not inhibit t he NF- κB activation in T47D cells. However, this result should be confirm ed by experim ent in WiDr cells.
The m echanism of apopt osis induct ion of PGV- 0 also involves up regulat ion of pro- apopt ot ic prot ein Bax, alt hough t his effect is not bet t er t han curcum in. Moreover, PGV- 0 also induces apopt osis by suppression of COX 2 expression by w hich it up regulat es p53 t ranscript ion fact or. Mechanism s m ediat ed t his response need t o be explored. I nt erest ingly, PGV- 1 did not show sim ilar response com pare t o curcum in and PGV- 0 alt hough it gave t he st rongest growt h inhibit ory effect and showed t he bet t er ant i- inflam m at ory effect t han curcum in in vivo ( Molnas Team , 2001) . PGV- 1 seem s t o induce apopt osis t rough t he inhibit ion of NF- κB-dependent anti-apoptotic genes. The present st udy show ed t hat PGV- 1 significant ly inhibit t he
κB in T47D cells. Since PGV-1 did not induce the act ivat ion of NF- expression of pro- apopt ot ic Bax, it s pro- apopt ot ic effect could be
Proceeding Molecular Targeted Therapy Symposium 2008
53 Endah Puji Septisetyani, et al.
κB/ Rel A anti-apoptotic downstream such t rough t he inhibit ion of NF- as Bcl- xL, Bcl- 2, or X- linked inhibit or of apopt osis ( XI AP) .
I n T47D cells, PGV- 1 t reat m ent inhibit s cell cycle progression at G2/ M and m it osis phase ( Da’i, 2007) . Dist urbance on m icrot ubule and failure of m it osis com plet ion result in act ivat ion of m it ot ic checkpoint indicat ed by t he expression of p21 by p53- independent in T47D cells. I t caused accum ulat ion of cells at G2/ M phase as w ell as hyperploid cells. These accum ulat ions subsequent ly induce cells t o undergo apopt osis ( Wang et al., 2000) . Furt herm ore, it needs t o be exam ined whet her PGV- 1 also inhibit s cell growt h t rough cell cycle arrest in WiDr cells by accum ulat ion of cells in G2/ M phase.
Figure 4. I nduct ion of Bax expression in WiDr cells. ( A) Cont rol cell wit hout
μM Bax ant ibody, ( B) cont rol cells, ( C) cells t reat ed wit h 30 μM PGV-0, and (E) cells curcum in, ( D) cells t reat ed wit h 50 μM PGV-1. The intensity of brown color of t reat ed wit h 10 cyt oplasm indicat es t he level of proapopt ot ic prot ein Bax expression, t he m ore t he int ensit y t he higher Bax expression.Apoptosis Mediated Cytotoxicity.............
Figure 5. I nhibit ion of COX 2 expression in WiDr cells. ( A) Cont rol cells
wit hout COX 2 ant ibody, ( B) cells t reat ed wit h 30 μM curcum in, ( C) cells t reat ed wit h 50 μM PGV-0, (D) control cell, (E) cells t reat ed wit h 10 μM PGV-1. Blue colour of cytoplasm indicates inhibit ion of COX 2 expression and brown color of cyt oplasm indicat es COX 2 expression, t he m ore t he int ensit y t he higher COX 2 expressionConc lusion
Taken t oget her, all of t hose result s indicat e t hat PGV- 1 is t he m ost prospect ive chem oprevent ive agent alt hough PGV- 1 act s via independent inhibit ion of COX 2 expression pat hway. PGV- 1 has t he st rongest effect on apopt osis induct ion in WiDr cells. Moreover, t he m olecular m echanism of grow t h inhibit ion and apopt osis induct ion of PGV- 1 is im port ant t o be explored.
Ac k now le dge m e nt
We t hank Prof Kaw aichi NAI ST- Japan for t he kindly pr oviding plasm id and m at erials for gene expression experim ent .
Proceeding Molecular Targeted Therapy Symposium 2008
55 Endah Puji Septisetyani, et al.
Re fe re nc e s
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