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: edy.meiyanto@gmail.com
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 apopt osis. Tw o analogues of curcum in, PGV- 0 ( 2,5-bis- ( 4′- hydroxy- 3′ -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 of PGV- 0 and PGV- 1 in WiDr cell det erm ined by MTT assay were 45 μM and 8 μM, respect ively. Moreover, evidence of apopt osis 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
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 curcum in, PGV- 0 ( 2,5-bis- ( 4′- hydroxy- 3′- m et hoxy) - benzilidine-cyclopent anone) and PGV- 1 ( 2,5-bis- ( 4′- hydroxy,3′,5′ dim et hyl) -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
Pla sm id
The kB- responsive luciferase report er const ruct and t he
NF-κB expression vect or, pCMX-lacZ, w as a kind gift from Prof. Kaw aichi, 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 ( 3x103 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 . 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
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 t ransfect ion cell were t reat ed w it h m edium cont aining 1.5 μM PGV- 1, 20 μM curcum in, 15 μM PGV- 0 and 1 ng/ m l of TNF-α. 24 hours aft er 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 C50) 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 C50 values of PGV- 0, PGV- 1, and
curcum in were 45, 8, and 27 μM, respect ively. PGV- 1 exhibit ed t he 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 C50
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 cell. ( A) cont rol cells, ( B) , ( C) , and ( D) cell t reat ed wit h 30 μM
curcum in, 50 μM PGV- 0, and 10 μM PGV- 1 at incubat ion t im e 15 h.
I 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-κ
Figure 3. Suppression on NF- B act ivat ion in T47D cells. ( A) cont rol cell
induced by TNF-α, ( B) , ( C) , ( D) cells t reat ed wit h 20 μM
curcum in, 1.5 μM PGV- 1, 10 μM PGV- 0, ( E) cont rol cell w it hout
TNF- α. PGV- 1 showed t he st rongest suppr ession on NF-κB
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 expression, NF-κB dependent t ranscript ional act ivit y, and expression of NF-κB- dependent ant i- apopt ot ic 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 NF-κB independent m echanism ( Collet and Cam pbell, 2006) . We hypot hesized t hat t he curcum in analogue PGV- 0 induces apopt osis t rough independent NF-κB / Rel A t ranscript ional act ivit y since PGV- 0 did not inhibit t he NF-κB act ivat ion in T47D cells. How ever, t his result should be confirm ed by experim ent in WiDr cells.
t rough t he inhibit ion of NF-κB/ Rel A ant i- apopt ot ic downst ream such 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
Bax ant ibody, ( B) cont rol cells, ( C) cells t reat ed wit h 30 μM
curcum in, ( D) cells t reat ed wit h 50 μM PGV- 0, and ( E) cells
t reat ed wit h 10 μM PGV- 1. The int ensit y of brow n color of
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) cont rol cell, ( E) cells
t reat ed wit h 10 μM PGV- 1. Blue colour of cyt oplasm indicat es
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 expression
Conc 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
Re fe re nc e s
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