Research ArticleInduction of Apoptosis of 2410158406-Trihydroxybenzophenonein HT-29 Colon Carcinoma Cell Line

Ma Ma Lay1 Saiful Anuar Karsani12 and Sri Nurestri Abd Malek1

1 Institute of Biological Sciences Faculty of Science University of Malaya 50603 Kuala Lumpur Malaysia2 University of Malaya Centre for Proteomics Research (UMCPR) University of Malaya 50603 Kuala Lumpur Malaysia

Correspondence should be addressed to Ma Ma Lay mamalaystergmailcom

Received 24 August 2013 Accepted 22 October 2013 Published 22 January 2014

Academic Editor Bruno C Cavalcanti

Copyright copy 2014 Ma Ma Lay et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

2410158406-Trihydroxy-4-methoxybenzophenone was isolated from the ethyl acetate fraction of Phaleria macrocarpa (Scheff) Boerlfruits It was found to inhibit cell proliferation in HT-29 human colon carcinoma cell line but caused little damage to WRL-68normal human liver and MRC-5 normal human fibroblast lung cell lines The compound was found to sharply affect the viabilityof HT-29 cells in a dose- and time-dependent manner HT-29 cells treated with the compound showed morphological changesunder microscopic examination such as cell shrinkage membrane blebbing DNA fragmentation and the occurrence of apoptoticnucleiThe percentage of early apoptotic late apoptotic and dead or necrotic cells was determined by flow cytometry using annexinV-FTICPI staining In addition flow cytometry showed that when the HT-29 cells were treated with 115120583M of the compoundit resulted in G

0G1phase arrest in a time-dependent manner Western blot revealed an upregulation of PUMA Bak Bcl-2 and

Mcl-1 proteins suggesting that the compound induced apoptosis in HT-29 cells by regulating these proteins

1 Introduction

Cancer refers to a family of diseases in which tissues multiplyand spread unregulated throughout the body the result ofwhich may lead to death [1] It is a major public healthproblem and has significantly increased the worldwide deathrate One of themost common types of cancer is colon cancerModern advances in cellular and molecular biology haveincreased our understanding of the various mechanisms ofcancer [2] Nowadays there are many natural compoundsand mechanism-based approaches to cancer treatment thathave the potential to be successful The development ofanticancer drugs has also seen a massive contribution fromnatural products [2]

Phaleria macrocarpa (Scheff) Boerl is a medicinal plantbelonging to the Thymelaeaceae family and in Indonesia itis locally known as Mahkota Dewa The edible fruits of thisplant are usuallymixedwith other Indonesian herbs and usedin herbal medicine [3] The fruits of P macrocarpa consist ofthe skin which have a bright red color a white fibrous andwatery flesh a shell and a seed In alternative medicine it

is used in the treatment of cancer diabetes mellitus nervepain kidney failure and disorder liver dysfunction seriousillnesses hypertension skin diseases and management ofcholesterol levels

Research on the use of the extracts of P macrocarpa fruitsas a possible anticancer agent is extensive Prior research hasshown that an ethanol extract from the flesh of the P macro-carpa fruit toxic towards HeLa cell line derived from cervicalcarcinoma [4] It has been shown that different parts of Pmacrocarpa fruit showed excellent antioxidant activities andgood anti-inflammatory and high cytotoxic activities againstHT-29 MCF-7 and HeLa cell lines [5] Gallic acid isolatedfrom P Macrocarpa has been shown to possess anticancerproperties against human esophageal cancer cells (TE-2) butno cytotoxic effect was observed on noncancerous (CHEK-1) cells [6] The major compound of Phaleria obtained fromP macrocarpa fruits has been found to have high cytotoxiceffect on MDA-MB231 breast cancer cell lines [7] DLBS1425of the extraction of P macrocarpa flesh fruits inhibitedproliferation of MDA-MB231 and MCF-7 cells activationof caspase 9 and downregulation of Bax and Bcl-2 at the

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 468157 12 pageshttpdxdoiorg1011552014468157

2 BioMed Research International

Phaleria macrocarpa (Scheff) Boerl fruits

Dried and ground plant materialExtraction with methanol (three times)

Methanol extractFractionation with hexane

Hexane-soluble fraction Hexane-insoluble fraction

Water fraction Ethyl acetate-soluble fraction (582 g)

Column chromatography with silica gelEluting solvent system is hexane and ethyl acetate

Mixture withstigmast-4-en-

one Palmitic acid(712mg)

(667mg)

Washed with chloroform

FF1 FF2 FF3 FF4 FF5 FF6 FF7 FF8 FF9

120573-Sitosterol

Partitioning (vv) with ethyl acetate water (1 1)

24998400 6-Trithydroxy-4-methoxybenzophenone (2380mg)

Figure 1 Extraction and isolation of bioactive compounds from ethyl acetate fraction of Phaleria macrocarpa (Scheff) Boerl fruits usingsilica gel column chromatography

mRNA level [8] In addition it was also revealed that 2610158404-trihydroxy-4-methoxybenzophenone and 410158406-dihydroxy-4-methoxybenzophenone 2-o-120573-D-glucopyranoside isolatedfrom the fruits displayed antiproliferation activity againstthe breast cancer cell line (MDA-MB231) and the forma-tion of DNA fragments as well as decreasing Bcl2 mRNAexpression increasing Bax mRNA expression and apop-tosis inducer activity [9] Furthermore 2641015840-trihyroxy-4-methoxybenzophenone from an ethyl acetate extract of theleaves of P macrocarpa possessed good antioxidant activity[10]

In our present study the bioactive 2610158404-trihydroxy-4-methoxybenzophenone was isolated from the ethyl acetateextract of the P macrocarpa fruits and analyzed using gas-chromatography mass spectrometry (GC-MS) and nuclearmagnetic resonance which were 1H 13C DEPT HMQCand HMBC The HT-29 human colon cancer cell line wasthen selected for evaluation of bioactivity and molecularmechanisms to examine the efficiency of 2610158404-trihydroxy-4-methoxybenzophenone

2 Materials and Methods

21 Extraction and Isolation of Bioactive Compounds Pmacrocarpa fruits were obtained from Yogyakarta Indonesia(July 2009) The fruits (1 kg) were cut washed air-driedand crushed The powdered fruits were extracted with 70methanol for three days at room temperature The resultingsolutionwas then filtered combined and concentrated underreduced pressure at 37∘C using a rotary evaporator to yielda crude methanol extract The methanol extract was then

dissolved in hexane and this produced hexane soluble andinsoluble fractionsThehexane soluble fractionwas separatedwith water and an ethyl acetate solution (1 1) to yield waterfractions and ethyl acetate fractions The dried residue of thefour extracts was then subjected to bioassays The bioactiveethyl acetate fraction was subjected to column chromatog-raphy for isolation of bioactive compounds The method forthe extraction fractions and column chromatography of thefruits is summarized in Figure 1

22 Analysis of Bioactive Compounds

221 Gas Chromatography-Mass Spectrophotometry (GC-MS) GC-MS was performed as previously described [11]

222 Nuclear Magnetic Resonance (NMR) The NMR anal-ysis was carried out on a JEOL 400MHz FT-NMR (JEOLJapan) with TMS (tetramethylsilane) as the internal standardSpectra were obtained from Proton 1H-NMR (400MHz)carbon 13C-NMR (400MHz) HMBC and HMQC analysis

23 Preparation of Test Compound (2410158406-Trihydroxy-4-methoxybenzophenone) The test compound used was 2410158406-trihydroxy-4-methoxybenzophenone isolated from Pmac-rocarpa Stock solutions of 2410158406-trihydroxy-4-methox-ybenzophenone were prepared at a concentration of 38mMwith absolute DMSO solution The test sample was thenprepared through serial dilution of the stock solution to giveconcentrations of 4120583M 38 120583M 96 120583M 192 120583M and 384 120583MThe final concentration of DMSO in each treatment well wasnot in excess of 05 DMSO

BioMed Research International 3

24 Cytotoxicity Screening for Bioactive Compounds

241 Cell Culture The HT-29 human colon carcinoma cellline WRL-68 normal liver cells and MRC-5 normal lungfibroblast carcinoma cells were purchased from the Ameri-can Type Culture Collection (ATCC USA) The cells wereplaced into tissue culture flasks and grown in a mediumcontaining 10 fetal bovine serum 100 120583gmL of peni-cillinstreptomycin and 100 120583gmL of amphotericin B at 37∘Cin a humidified atmosphere containing 5 CO

2

242MTTCell Proliferation Assay TheMTT (345-dimeth-ylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) cell pro-liferation assay was based on the protocol described byMosmann [12] Briefly cells from a confluent tissue cultureflaskwere spun at 1000 rpm for fiveminutes and resuspendedin 10mL of growth medium Cells were then seeded into96 well plates and incubated in a CO

2incubator at 37∘C for

24 hours to allow the cells to adhere and achieve 70ndash80confluence After 24 hours the media was removed and thetest compound at varying concentrations of 4 38 96 192and 384 120583M with 200120583L of 10 medium was added to therespective wells For negative control untreated cells wereused After 24 h 48 h and 72 h of incubation respectivelyeach well had 10 120583L of the MTT stock solution added to thetest sample and was then incubated in a dark place for threehoursThemediumwith theMTT solution was removed and200120583L of absolute DMSO was added to each well in orderto solubilise the formazan crystals that had formedThe totalamount of formazan crystals was examined bymeasuring theabsorbance at 540 nm using an ELISA plate reader The assaywas carried out in triplicate

The percentage of inhibition () was calculated using thefollowing formula

percentage of inhibition ()

=OD control minusOD sample times 100

OD control

(1)

where OD = optical densitiesThe IC

50value is the concentration of test compounds

which cause 50 inhibition or cell death average from threeexperiments and was obtained by plotting the percentage ofinhibition versus the concentration of test compounds [13]

25 Morphological Detection of Apoptosis

251 Inverted and Phase-Contrast Microscopy HT-29 cellswere placed in a Petri dish (30mm) at a density of 1 times 105cellswell plate and grown for 24 h The test compound wasthen added at IC

50value and the cells were further incubated

for 24 h 48 h and 72 h respectively After the various incuba-tion periods morphological changes in the apoptotic bodiesof the HT-29 cells were examined by inverted and phase-contrast microscopy (Leica Germany) and photographed

252 Fluorescence Microscopy HT-29 cells were placed ina Petri dish (30mm) at a density of 1 times 105 cellswell

plate and grown for 24 h The test compound (115 120583M) wasadded to each dish and the cells were further incubated for72 h After the incubation period the cells were detachedwith 025 accutase in phosphate buffer saline (PBS) thesupernatant was discarded and the cells were resuspendedin 1mL of PBS Cells (100 120583L) were then incubated with 5120583Lof acridine orange (AO) and 5 120583L of propidium iodide (PI)for 10min at room temperature in the dark Stained cells(10 120583L) were put in three wells of the coated glass slideswith 20120583L of mounting media and stored at minus20∘C prior toanalysis These double stained cells were photographed usinga fluorescencemicroscope (Leica Germany) All experimentswere performed in triplicate

26 Annexin V Staining Assay The quantification of celldeath was determined by flow cytometry using the AnnexinV-FITC apoptosis detection kit according to the manufac-turerrsquos instructions (BD Pharmingen BD Bioscience USA)Briefly 1 times 106 of the HT-29 cells were seeded into eachPetri dish (30mm) and after a 24 h incubation various con-centrations of the test compound were added and incubatedfor 24 h 48 h and 72 h respectively The cells were thenwashedwith PBS suspended in annexinV binding buffer andthen added to an annexin V-FITC solution and propidiumiodide (PI) for 10 minutes at room temperature The sampleswere then analyzed using FACScalibur (BD Bioscience USA)using CellQuest Pro analysis software (Becton DickinsonUSA)

27 Cell Cycle Analysis HT-29 cells were placed in Petridishes at a density of 1 times 105 cellswell and grown for 24 hand then the cells were treated with 2410158406-trihydroxy-4-methoxybenzophenone (at IC

50) at 37∘C for 24 h 48 h and

72 h After incubation the cells were collected by trypsiniza-tion fixed in ethanol washed in warm PBS and incubatedat minus20∘C overnight Next the cells were washed with 1mLof cold PBS and stained with propidium iodide according tothe Cycle TEST PLUS DNA Reagent Kit (Becton DickinsonUSA) protocol The cells were sorted in a FACScalibur flowcytometer (BD USA) using CellQuest Pro software (BectonDickinson USA) and a quantitative analysis of the cellcycle distribution was performed using a trial version of theModFit LT software version 40

28 Western Blot Analysis The HT-29 cells were culturedin 25 cm3 tissue culture flasks and incubated at 37∘C under5 CO

2until confluent The cells were then treated with a

double concentration of IC50value for the 2410158406-trihydroxy-

4-methoxybenzophenone for 24 h 48 h and 72 h respec-tively After incubation at the various times the cells weredetached with accutase in PBS washed thoroughly withPBS twice and were then lysed with protein lysis buffer(Sigma USA) containingprotease inhibitors and shaken byhand for 15min at room temperature The lysates were thencentrifuged at 13000timesg for 20min at 4∘Cand the supernatantwas collected for protein samples and kept at 40∘C until SDSpage analysis Protein concentration was determined using

4 BioMed Research International

a BCA (bicinchoninic acid) protein assay kit (InvitrogenUSA) with bovine serum albumin as the standard

The proteins were transferred into polyvinylidene diflu-oride (PVDF) membranes by semidry transfer Bands werevisualized using the Western Max Horseradish PeroxidaseChromogenic Detection Kit protocol (AMRESCO USA)Briefly the membranes were blocked in a blocking bufferfor 30min at room temperature Primary antibodies forBcl-2 Bcl-xL PUMA Mcl-1 Bak Bad and Bax were thenadded The membranes were incubated with gentle agita-tion at room temperature overnight After incubation themembranes were washed with washing buffer two timesand then incubated with Western Max HRP ConjugatedAnti-Rabbit Antibody for 45min at room temperature withgentle agitation After discarding the secondary antibodysolution the membranes were washed twice and incubatedwith DAB substrate solution containing hydrogen peroxideat room temperature until a brown color developed Finallythe membranes were dried photographed and stored in thedark

29 Data Analysis The numbers of viable cells were countedusing a haemocytometer with trypan blue exclusion MTTtest was performed in triplicate The data of the cytotoxicityassay was expressed as the mean plusmn standard derivation (SD)The mean plusmn standard derivations (SD) in each cell cyclephase were also calculated for the annexin V-FITCPI doublestaining assayThe results from treated and untreated controlcells were analyzed using Studentrsquos 119905-test Differences with a119875 value of gt005 as determined using version 160 of SPSSwere considered statistically significant

3 Results and Discussions

31 Extraction and Isolation The yield of dark brownmethanol extract (472 g 47) hexane fraction (210 g020) ethyl acetate fraction (122 g 122) and waterfraction (298 g 290) were determined The ethyl acetatefraction (98 g) was subjected to silica gel column chromatog-raphy on a Merck Kieselgel 60 (400 g 0063ndash0200mmmeshsize) initial elution with hexane followed by ethyl acetateenriched with increasing percentages of acetone and mon-itored with TLC to give several main fractions FF1 (07 g)FF2 (079 g) FF3 (042 g) FF4 (07 g) FF5 (05 g) FF6 (08)FF7 (15 g) FF8 (18 g) and FF9 (23 g) The components inthese fractionswere identified usingGC-MSNMR techniqueand some fractions were purified by recrystallization andseparation with specific solvents

GC-MS analysis of the ethyl acetate fraction of the Pmacrocarpa showed the presence of phenol (mz 94) 26-dimethoxy phenol (mz 154) 2-methoxy phenol (mz 124)120573-sitosterol (mz 414) stigmast-4-en-3-one (mz 412) flamenol(mz 140) palmitic acid (mz 256) methyl palmitate oleicacid (mz 264) and other unknown components

A mixture with stigmast-4-en-3-one (mz 276) wasdetected in (FF 1) 120573-Sitosterol (mz 414) was obtained fromfraction 5 (FF 5) and palmitic acid was also obtained fromfraction 2 (FF2) by identification GC-MS analysis The pure

bioactive compoundwas obtained from fraction 4 (FF 4) afterrecrystallization This compound was identified as 2 41015840 6-trihydroxy-4-methoxybenzophenone using NMR and GC-MS analysis

32 Identification of Isolated Bioactive Compound 2410158406-Trihydroxy-4-methoxybenzophenone or 26-Dihydroxy-4-methoxyphenyl 4-Hydroxyphenyl methanone The structureof this compound was established on the basis of EI-MSdata together with 1H and 13C NMR spectra as shown inFigure 2(a) The peak at retention time 4393min in thetotal ion chromatogram of fraction FF 4 gave a molecularion peak at mz 260 in the mass spectrum which wasconsistent with the molecular weight of 2410158406-trihydroxy-4-methoxybenzophenone having a molecular formula ofC14H12O5 Fragment ions resulting from cleavages on either

side of the carbonyl group were observed atmz = 121 and 93respectively as shown in Figure 3(a) The formation of otherfragment ions is explained in Figure 2(b)

The two pairs of equivalent aromatic hydrogen namelyH21015840 H61015840 and H

31015840 H51015840 in ring B and a pair of equivalent

aromatic hydrogens in ring A H3H5 are visible at 120575 759 (d)

675 (d) and 595 (s) respectively in the 1H NMR spectrum(CDCl

3CD3OD 400MHz) (Table 1) The corresponding

three equivalent pairs of aromatic carbons C21015840 C61015840 C31015840 C51015840

and C3C5 can also be seen at 120575 13405 11658 and 9535

in the 13C NMR spectrum (CDCl3CD3OD 100MHz) in

Figure 2(a) The signals also showed an aromatic methoxygroup at 120575 374 and 5586 in 1H and 13C NMR spectrarespectively The ketone carbonyl signal appears at 120575 19959Along with the chemical shifts of the remaining carbons theassignment of all the NMR signals is summarized in Table 1and Figure 2(a) The assignment of 1H and 13C NMR signalsis also summarized together with the 1H-13C HMBC andHMQC data in Table 1 as well as in Figures 2(c) and 2(d)

33 Cytotoxicity Screening UsingMTT Cell Proliferation Assay

331 Screening for Cytotoxic Activity of 2410158406-Trihydroxy-4-methoxybenzophenone Cell proliferation is an importantmechanism for the growth development and regeneration ofeukaryotic organisms but it is nevertheless also the primarycause of some of the most debilitating diseases one of whichis cancer [14]

In this study the cytotoxicity screening of the 2410158406-trihydroxy-4-methoxybenzophenone against the HT-29human colon carcinoma cell line was performed using theMTT cell proliferation assay and the cell viability of HT-29cells was measured using the trypan blue exclusion assayThe HT-29 cells were treated with various concentrations (438 96 192 and 384 120583M) for 24 h 48 h and 72 h respectivelyThe results showed that inhibition of cell growth significantlyincreased in a dose-dependent and time-dependent mannerwhen the HT-29 cells were treated with 2410158406-trihydroxy-4-methoxybenzophenone at 4 38 96 192 and 384 120583M for 24 h48 h and 72 h except for 96 120583M for 24 h as shown in Figures3(b) and 4(a) The IC

50values of the compound against the

HT-29 cells were 172 plusmn 221 144 plusmn 266 and 122 plusmn 169 120583M

BioMed Research International 5

374(5586)

9535595

(16216) OH OH

OH O

O 675

(11658)

759(13405)

(13377)

(16407)

(11658)675

759(13405)

(19959)(10905)

(16216)

595

(9535)

(16658)

1

23

45

6

1998400

29984003998400

4998400

5998400

6998400

A

(a)

HCH2

OH OH

OH

O

O

minusCO

minusCO

minusCO

minusCOminusCH3 minusH

minusH

mz 260

259

95 138 166 167

64

65

93121

∙

∙∙

+ ∙

(b)

(c) (d)

Figure 2 (a) Structure of 2410158406-trihydroxy-4-methoxybenzophenone showing assignment of protons and carbons (b) Proposedfragmentation of 2410158406-trihydroxy-4-methoxybenzophenone (c) HMBC analysis of isolated 2410158406-trihydroxy-4-methoxybenzophenone(d) HMQC analysis of isolated 2410158406-trihydroxy-4-methoxybenzophenone

for 24 h 48 h and 72 h In contrast the 2410158406-trihydroxy-4-methoxybenzophenone compound displayed mild cytotoxiceffect on human normal liver cells (WRL-68) with IC

50

values of ge 384 plusmn 102 331 plusmn 069 and 235 plusmn 103 120583M for24 h 48 h and 72 h respectively as shown in Figure 3(d)and human normal fibroblast lung cells (MRC-5) with IC

50

values of ge 384 plusmn 100 269 plusmn 096 and 356 plusmn 121 120583M for24 h 48 h and 72 h respectively as shown in Figure 3(c)

34 Morphological Studies HT-29 cells were treated with theisolated bioactive compound at IC

50concentrations (115 120583M)

for incubation periods of 24 h 48 h and 72 h respectivelyAfter these incubation periods the morphological changesin the cells were examined using inverted and phase-contrastmicroscopy The cells displayed changes that are known tobe associated to apoptosis including membrane blebbingcell shrinkage chromatin condensation apoptotic nuclei andDNA fragmentation (Figures 4(a) and 4(b))

35 FluorescenceMicroscopy Thecells were observed under afluorescencemicroscope at different excitations after stainingwith acridine orange and propidium iodide (AOPI) fornuclei containing DNA The results showed that the viablecells revealed the green nuclei dead cells and late apoptoticand necrotic cells displayed red nuclei and early apoptotic

cells indicated orange nuclei in Figure 4(c) Thus AOPIstaining of HT-29 showed that the cells had undergoneremarkable morphological changes in apoptotic bodies

36 Annexin V Staining Assay The most important molec-ular mechanism used in the treatment of anticancer drugs isprogrammed cell death or apoptosis in the chemotherapeuticapproach [15 16] In our study staining with Annexin V-FITC and propidium iodide (PI) may distinguish betweenintact cells early apoptosis late apoptosis or cell death [17]We also used single staining with Annexin V-FITC or PI todistinguish between live cells and dead cells however singlestaining could not distinguish between early apoptotic cellslate apoptotic cells or dead cells or necrotic and live cells

Cells were treated with various concentrations (96 120583M192 120583M and 288 120583M) of the compound for 24 h 48 h and72 h respectively They were then harvested with accutaseand phosphate buffer saline and centrifuged after which theywere stained with Annexin V-FITC and propidium iodideand analyzed in FACScalibur with CellQuest Pro softwareThe number of early apoptotic cells late apoptotic cells livecells and necrotic cells was counted in every 10000 cells ofeach treatment [18] All data was expressed as the mean plusmn SD(standard deviation) The standard deviation was calculatedfor the treated anduntreated cellsThe SPSS program (version

6 BioMed Research International

HCH2

OH OH

OH

O

O

minusCO

minusCO

minusCO minusCOminusCH3 minusH

mz

260

259

95 138 166 167

64

65

93121

∙∙

∙

minusH∙

∙

10 30 50 70 90 110 130 150 170 190 210 230 250 270

9000

8000

7000

6000

5000

4000

3000

2000

1000

0

259

243231216207199187175

166

138

155147130

121

110

102

93

8677

65

53

Abun

danc

e

mz

(a)

0102030405060708090

100

1 10 25 50 100

Inhi

bitio

n (

)

Concentration (120583M)

24h48h72h

HT-29

(b)

Inhi

bitio

n (

)

Concentration (120583M)

24h48h72h

MRC-5

0102030405060708090

100

4 38 96 192 384

(c)

Inhi

bitio

n (

)

Concentration (120583M)

24h48h72h

WRL-68

0102030405060708090

100

4 38 96 192 384

(d)

Figure 3 (a) Mass-spectrum of gas chromatography mass spectrometry (GC-MS) analysis of 2410158406-trihydroxy-4-methoxybenzophenone(b) In vitro cytotoxic effects of 2410158406-trihydroxy-4-methoxybenzophenone on (b) HT-29 colon cancer cells (c) MRC-5 normal lung cellsand (d) WRL-68 normal fibroblast cells Cells were treated with various concentrations of 2461015840-trihydroxy-4-methoxybenzophenone fromethyl acetate fraction of Phaleria macrocarpa (Scheff) Boerl fruits for 24 h 48 h and 72 h prior to the determination of cytotoxicity by MTTcell proliferation assay Each value is expressed as mean plusmn standard deviation of three measurements

160) found a significant difference between the treated anduntreated cells (119875 gt 005)

In flow cytometry analysis of Annexin V-FITCPI doublestaining the late or secondary necrotic apoptotic cells werevisible in the upper right (UR) and early apoptotic cells inthe lower right (LR) quadrants while the primary necroticcells were visible in the upper left (UL) and live cells inthe lower left (LL) quadrants respectively In untreated HT-29 cells 9228 of the cells were viable 185 was in earlyapoptosis and 321 cells were in late apoptosis or secondarynecrotic stage When the HT-29 cells were treated with

96 120583M 192 120583M and 288120583M of the compound for 24 h theviable cells were 6926 3516 and 2383 the primarynecrotic cells were 1549 3040 and 1963 the earlyapoptotic cells were 296 846 and 671 and the lateapoptotic cells were 1228 2598 and 5081 respectivelyAfter 48 h incubation live cells were at 4961 3422 and2680 primary necrotic cells were at 804 3505 and1002 early apoptotic cells were at 1087 474 and 851and late apoptotic cells were at 3148 2555 and 5467respectively After 72 h incubation live cells were at 52211273 and 5464 primary necrotic cells were at 3069

BioMed Research International 7

Table 1 1H NMR (CDCl3CD3OD 400MHz) 13C NMR (CDCl3CD3OD 100MHz) HMQC and HMBC data of isolated 2410158406-trihydroxy-4-methoxybenzophenone or 26-dihydroxy-4-methoxyphenyl 4-hydroxyphenyl methanone

Positiongroup 120575C 120575H (mult J in Hz) 1H-13C HMBC HMQC1 10905 mdash mdash mdash26 16216 mdash mdash mdash35 9535 595 (s) C-1 C-2C-6 C-4 C-3C-54 16658 mdash mdash mdash11015840 13377 mdash mdash mdash2101584061015840 13405 759 (d 88) C-11015840 C-31015840C-51015840 C-41015840 C=O C-21015840C-61015840

3101584051015840 11658 675 (d 88) C-11015840 C-21015840C-61015840 C-41015840 C-31015840C-51015840

41015840 16407 mdash mdash mdashC=O 19959 mdash mdash mdashOCH3 5586 374 (s) C-4 COCH3

Control 24h 48h 72h

(a)

Control 24h 48h 72h

(b)

Live cells Necrotic and dead cellsApoptotic cells

(c)

Figure 4 (a) Cells were treated with compound (30 120583M) for 24 h 48 h and 72 h which induced morphological changes typical of apoptosisin HT-29 colon carcinoma cells Control or treated cells were observed under inverted microscope and photographed (b) Control or treatedcells were observed under phase contrast microscope and photographed (c) Treatment with IC

50value (30 120583M) of 2410158406-trihydroxy-4-

methoxybenzophenone for 48 h induces morphological changes typical of apoptosis in HT-29 colon cancer cells After being stained withacridine orange and propidium iodide treated cells were observed under fluorescencemicroscopymdashlive cells stained (green colour) apoptoticcells (orange) and necrotic cells or dead cells (red)

8 BioMed Research International

1396 and 3663 early apoptotic cells were at 133 1434and 028 and late apoptotic or secondary necrotic cells wereat 1577 5897 and 845 respectively All these data areshown in Figures 5(a) and 5(b)

The results indicated that the effects of programmed celldeath or apoptosis on HT-29 colon carcinoma cells wereinduced by 2410158406-trihydroxy-4-methoxybenzophenone in atime-and dose-dependent manner

37 Cell Cycle Analysis One of the most important mecha-nisms in anticancer drug treatment is cell cycle arrest whichcan be measured by DNA content DNA (deoxyribonucleicacid) plays an essential role in cell reproduction and cell lifeand death and also carries genetic information for all livingorganisms and consists of two sets of chromosomes [15 16]When 2NDNA is present in the G

0andG

1phases of each cell

cycle the G2and M phases in the cell cycle are presented by

4N DNA which has a double 2N number of chromosomeswhile the S-phase synthesizes DNA replication using flowcytometry analysis

The HT-29 cells were treated with isolated bioactive2410158406-trihydroxy-4-methoxybenzophenone for 24 h 48 hand 72 h Treated and untreated cells were harvested withaccutase and phosphate buffer saline centrifuged andstained with propidium iodide The HT-29 cells were thenanalyzed by flow cytometry using CellQuest Pro software andtheir distribution in different phases of the cell cycle is shownin Figure 6(a) The resulting data were tabulated using thetrial version of the ModFit LV 40 software to represent apercentage of each cell cycle phase (G

0G1 S and G

2M) as

shown in Figure 6(b)Treatment with the 2410158406-trihydroxy-4-methoxyben-

zophenone compound (at IC50

value of 115 120583M) resulted incell cycle arrest in the G

0G1phase in a time-dependent

manner Standard deviationwas calculated for the treated anduntreated cellsThe significance of the difference between thetreated and untreated cells in each cell cycle phase namelyG0G1 S and G

2M was determined by Studentrsquos 119905-test and

the 119875 value was gt005 using SPSS version 160After incubation for 24 h 48 h and 72 h the percentage

of diploid cells in the G0G1phase progressively increased

to 7069 7998 and 8821 and the cell percentage inthe G

2M and S phases was 1361 317 and 320 and

1570 1685 and 860 respectively (Figure 6(b)) Theaneuploid cells in the G

0G1and S phases were not stable

because of abnormal chromosome problems in the centrioleswhere extra or missing chromosomes occurred

Every diploid cell has two sets of chromosomes (2N)which occur in the G

0G1phase of each cell cycle and

tetraploid cells have double the normal 2N number of chro-mosomes (4N) which occur in the G

2M phase of each cell

cycle however aneuploidy refers to an abnormal number ofchromosomes which was observed as a significant differencebetween the G

0G1and G

2M phases in the flow cytometry

analysis We observed that the number of diploid cells andtetraploid cells in the G

0G1and S phases of each cell cycle

were heavily reliant on one another

Thus we have shown here that 2410158406-trihydroxy-4-methoxybenzophenone at 115120583M induced apoptosis in HT-29 cells during G

0G1arrest and the inhibition of cell growth

could be a result of the induction of apoptosis which may beresolved by cell cycle arrest which may consequently resultin programmed cell death

38 Western Blot Analysis Apoptosis or programmed celldeath consists of three major pathways the intrinsic pathwaythe extrinsic or death-receptor pathway and the signallingpathways that indicates death-associated proteolytic or nucle-olytic activities

The Bcl-2 family of proteins play an essential role in regu-lating the mitochondria-dependent pathway of apoptosis forhealth and diseases [19] Bcl-2 proteins are alternative targetsfor both pro- and antiapoptotic therapeutic approaches [20]There are two groups of proteins involved in apoptosismdashantiapoptotic or prosurvival proteins such as Bcl-2 Bcl-xlBcl-w Mcl-1 and A1 and proapoptotic proteins such as BaxBak PUMA Bok Bad Bid Bik Blk Hrk BNIP3 and BimLThe Bcl-2 family of proteins have been shown to regulateapoptosis in response to chemotherapy both in vivo and invitro [21 22] The Bcl-2 family of proteins are located inthe endoplasmic reticulummembrane the nuclear envelopeand in the mitochondrionrsquos outer membranes [23] The Bcl-2prosurvival proteins directly or indirectly regulate the releaseof cytochrome c from mitochondria

A member of the BH3 subgroup of the Bcl-2 family ofproteins is PUMA (p53-upregulated mediator of apoptosis)which interacts with antiapoptotic Bcl-2 family proteinssuch as Bcl-xL Bcl-2 Mcl-1 Bcl-w and A1 or proapoptoticproteins such as Bax and Bak It also involves p53-dependentand p53-independent apoptosis induced by different sig-nalling pathways Another antiapoptotic protein from theBcl-2 family is the myeloid cell leukemia sequence 1 (Mcl-1) which is involved in the upregulation of apoptosis andsurvival regulation and also plays a part in melanoma cellresistance towards Fas-mediated apoptosis Inhibiting themitochondrial signalling pathway of apoptosis can directlyaffect cancer therapy It is well known that Bcl-2 is a potentantiapoptotic protein which is expressed in HT-29 cellsIn our study the expression of apoptosis and cell cycleassociated proteins was determined using western blottingafter the HT-29 cells were treated with 2410158406-trihydroxy-4-methoxybenzophenone The expression of antiapoptotic(Bcl-2 and Mcl-1) and proapoptotic protein (PUMA andBak) in HT-29 colon human adenocarcinoma cells line wasexamined using the western blot technique To clarify themechanisms by which the compound induced apoptosis ofHT-29 cells the cells were incubated at an IC

50concentra-

tion (115 120583M) of 410158406-trihydroxy-4-methoxybenzophenonefor 24 h 48 h and 72 h Proteins were then extracted forwestern blotting As is shown in Figures 6(c) and 6(d) theexpression of Mcl-1 and PUMAwas significantly upregulatedwhen the HT-29 cells were treated with the compound for24 h 48 h and 72 hThe expression of Bcl-2 and Bak was only

BioMed Research International 7

Table 1 1H NMR (CDCl3CD3OD 400MHz) 13C NMR (CDCl3CD3OD 100MHz) HMQC and HMBC data of isolated 2410158406-trihydroxy-4-methoxybenzophenone or 26-dihydroxy-4-methoxyphenyl 4-hydroxyphenyl methanone

Positiongroup 120575C 120575H (mult J in Hz) 1H-13C HMBC HMQC1 10905 mdash mdash mdash26 16216 mdash mdash mdash35 9535 595 (s) C-1 C-2C-6 C-4 C-3C-54 16658 mdash mdash mdash11015840 13377 mdash mdash mdash2101584061015840 13405 759 (d 88) C-11015840 C-31015840C-51015840 C-41015840 C=O C-21015840C-61015840

3101584051015840 11658 675 (d 88) C-11015840 C-21015840C-61015840 C-41015840 C-31015840C-51015840

41015840 16407 mdash mdash mdashC=O 19959 mdash mdash mdashOCH3 5586 374 (s) C-4 COCH3

Control 24h 48h 72h

(a)

Control 24h 48h 72h

(b)

Live cells Necrotic and dead cellsApoptotic cells

(c)

Figure 4 (a) Cells were treated with compound (30 120583M) for 24 h 48 h and 72 h which induced morphological changes typical of apoptosisin HT-29 colon carcinoma cells Control or treated cells were observed under inverted microscope and photographed (b) Control or treatedcells were observed under phase contrast microscope and photographed (c) Treatment with IC

50value (30 120583M) of 2410158406-trihydroxy-4-

methoxybenzophenone for 48 h induces morphological changes typical of apoptosis in HT-29 colon cancer cells After being stained withacridine orange and propidium iodide treated cells were observed under fluorescencemicroscopymdashlive cells stained (green colour) apoptoticcells (orange) and necrotic cells or dead cells (red)

8 BioMed Research International

1396 and 3663 early apoptotic cells were at 133 1434and 028 and late apoptotic or secondary necrotic cells wereat 1577 5897 and 845 respectively All these data areshown in Figures 5(a) and 5(b)

The results indicated that the effects of programmed celldeath or apoptosis on HT-29 colon carcinoma cells wereinduced by 2410158406-trihydroxy-4-methoxybenzophenone in atime-and dose-dependent manner

37 Cell Cycle Analysis One of the most important mecha-nisms in anticancer drug treatment is cell cycle arrest whichcan be measured by DNA content DNA (deoxyribonucleicacid) plays an essential role in cell reproduction and cell lifeand death and also carries genetic information for all livingorganisms and consists of two sets of chromosomes [15 16]When 2NDNA is present in the G

0andG

1phases of each cell

cycle the G2and M phases in the cell cycle are presented by

4N DNA which has a double 2N number of chromosomeswhile the S-phase synthesizes DNA replication using flowcytometry analysis

The HT-29 cells were treated with isolated bioactive2410158406-trihydroxy-4-methoxybenzophenone for 24 h 48 hand 72 h Treated and untreated cells were harvested withaccutase and phosphate buffer saline centrifuged andstained with propidium iodide The HT-29 cells were thenanalyzed by flow cytometry using CellQuest Pro software andtheir distribution in different phases of the cell cycle is shownin Figure 6(a) The resulting data were tabulated using thetrial version of the ModFit LV 40 software to represent apercentage of each cell cycle phase (G

0G1 S and G

2M) as

shown in Figure 6(b)Treatment with the 2410158406-trihydroxy-4-methoxyben-

zophenone compound (at IC50

value of 115 120583M) resulted incell cycle arrest in the G

0G1phase in a time-dependent

manner Standard deviationwas calculated for the treated anduntreated cellsThe significance of the difference between thetreated and untreated cells in each cell cycle phase namelyG0G1 S and G

2M was determined by Studentrsquos 119905-test and

the 119875 value was gt005 using SPSS version 160After incubation for 24 h 48 h and 72 h the percentage

of diploid cells in the G0G1phase progressively increased

to 7069 7998 and 8821 and the cell percentage inthe G

2M and S phases was 1361 317 and 320 and

1570 1685 and 860 respectively (Figure 6(b)) Theaneuploid cells in the G

0G1and S phases were not stable

because of abnormal chromosome problems in the centrioleswhere extra or missing chromosomes occurred

Every diploid cell has two sets of chromosomes (2N)which occur in the G

0G1phase of each cell cycle and

tetraploid cells have double the normal 2N number of chro-mosomes (4N) which occur in the G

2M phase of each cell

cycle however aneuploidy refers to an abnormal number ofchromosomes which was observed as a significant differencebetween the G

0G1and G

2M phases in the flow cytometry

analysis We observed that the number of diploid cells andtetraploid cells in the G

0G1and S phases of each cell cycle

were heavily reliant on one another

Thus we have shown here that 2410158406-trihydroxy-4-methoxybenzophenone at 115120583M induced apoptosis in HT-29 cells during G

0G1arrest and the inhibition of cell growth

could be a result of the induction of apoptosis which may beresolved by cell cycle arrest which may consequently resultin programmed cell death

38 Western Blot Analysis Apoptosis or programmed celldeath consists of three major pathways the intrinsic pathwaythe extrinsic or death-receptor pathway and the signallingpathways that indicates death-associated proteolytic or nucle-olytic activities

The Bcl-2 family of proteins play an essential role in regu-lating the mitochondria-dependent pathway of apoptosis forhealth and diseases [19] Bcl-2 proteins are alternative targetsfor both pro- and antiapoptotic therapeutic approaches [20]There are two groups of proteins involved in apoptosismdashantiapoptotic or prosurvival proteins such as Bcl-2 Bcl-xlBcl-w Mcl-1 and A1 and proapoptotic proteins such as BaxBak PUMA Bok Bad Bid Bik Blk Hrk BNIP3 and BimLThe Bcl-2 family of proteins have been shown to regulateapoptosis in response to chemotherapy both in vivo and invitro [21 22] The Bcl-2 family of proteins are located inthe endoplasmic reticulummembrane the nuclear envelopeand in the mitochondrionrsquos outer membranes [23] The Bcl-2prosurvival proteins directly or indirectly regulate the releaseof cytochrome c from mitochondria

A member of the BH3 subgroup of the Bcl-2 family ofproteins is PUMA (p53-upregulated mediator of apoptosis)which interacts with antiapoptotic Bcl-2 family proteinssuch as Bcl-xL Bcl-2 Mcl-1 Bcl-w and A1 or proapoptoticproteins such as Bax and Bak It also involves p53-dependentand p53-independent apoptosis induced by different sig-nalling pathways Another antiapoptotic protein from theBcl-2 family is the myeloid cell leukemia sequence 1 (Mcl-1) which is involved in the upregulation of apoptosis andsurvival regulation and also plays a part in melanoma cellresistance towards Fas-mediated apoptosis Inhibiting themitochondrial signalling pathway of apoptosis can directlyaffect cancer therapy It is well known that Bcl-2 is a potentantiapoptotic protein which is expressed in HT-29 cellsIn our study the expression of apoptosis and cell cycleassociated proteins was determined using western blottingafter the HT-29 cells were treated with 2410158406-trihydroxy-4-methoxybenzophenone The expression of antiapoptotic(Bcl-2 and Mcl-1) and proapoptotic protein (PUMA andBak) in HT-29 colon human adenocarcinoma cells line wasexamined using the western blot technique To clarify themechanisms by which the compound induced apoptosis ofHT-29 cells the cells were incubated at an IC

50concentra-

tion (115 120583M) of 410158406-trihydroxy-4-methoxybenzophenonefor 24 h 48 h and 72 h Proteins were then extracted forwestern blotting As is shown in Figures 6(c) and 6(d) theexpression of Mcl-1 and PUMAwas significantly upregulatedwhen the HT-29 cells were treated with the compound for24 h 48 h and 72 hThe expression of Bcl-2 and Bak was only

8 BioMed Research International

1396 and 3663 early apoptotic cells were at 133 1434and 028 and late apoptotic or secondary necrotic cells wereat 1577 5897 and 845 respectively All these data areshown in Figures 5(a) and 5(b)

The results indicated that the effects of programmed celldeath or apoptosis on HT-29 colon carcinoma cells wereinduced by 2410158406-trihydroxy-4-methoxybenzophenone in atime-and dose-dependent manner

37 Cell Cycle Analysis One of the most important mecha-nisms in anticancer drug treatment is cell cycle arrest whichcan be measured by DNA content DNA (deoxyribonucleicacid) plays an essential role in cell reproduction and cell lifeand death and also carries genetic information for all livingorganisms and consists of two sets of chromosomes [15 16]When 2NDNA is present in the G

0andG

1phases of each cell

cycle the G2and M phases in the cell cycle are presented by

4N DNA which has a double 2N number of chromosomeswhile the S-phase synthesizes DNA replication using flowcytometry analysis

The HT-29 cells were treated with isolated bioactive2410158406-trihydroxy-4-methoxybenzophenone for 24 h 48 hand 72 h Treated and untreated cells were harvested withaccutase and phosphate buffer saline centrifuged andstained with propidium iodide The HT-29 cells were thenanalyzed by flow cytometry using CellQuest Pro software andtheir distribution in different phases of the cell cycle is shownin Figure 6(a) The resulting data were tabulated using thetrial version of the ModFit LV 40 software to represent apercentage of each cell cycle phase (G

0G1 S and G

2M) as

shown in Figure 6(b)Treatment with the 2410158406-trihydroxy-4-methoxyben-

zophenone compound (at IC50

value of 115 120583M) resulted incell cycle arrest in the G

0G1phase in a time-dependent

manner Standard deviationwas calculated for the treated anduntreated cellsThe significance of the difference between thetreated and untreated cells in each cell cycle phase namelyG0G1 S and G

2M was determined by Studentrsquos 119905-test and

the 119875 value was gt005 using SPSS version 160After incubation for 24 h 48 h and 72 h the percentage

of diploid cells in the G0G1phase progressively increased

to 7069 7998 and 8821 and the cell percentage inthe G

2M and S phases was 1361 317 and 320 and

1570 1685 and 860 respectively (Figure 6(b)) Theaneuploid cells in the G

0G1and S phases were not stable

because of abnormal chromosome problems in the centrioleswhere extra or missing chromosomes occurred

Every diploid cell has two sets of chromosomes (2N)which occur in the G

0G1phase of each cell cycle and

tetraploid cells have double the normal 2N number of chro-mosomes (4N) which occur in the G

2M phase of each cell

cycle however aneuploidy refers to an abnormal number ofchromosomes which was observed as a significant differencebetween the G

0G1and G

2M phases in the flow cytometry

analysis We observed that the number of diploid cells andtetraploid cells in the G

0G1and S phases of each cell cycle

were heavily reliant on one another

Thus we have shown here that 2410158406-trihydroxy-4-methoxybenzophenone at 115120583M induced apoptosis in HT-29 cells during G

0G1arrest and the inhibition of cell growth

could be a result of the induction of apoptosis which may beresolved by cell cycle arrest which may consequently resultin programmed cell death

38 Western Blot Analysis Apoptosis or programmed celldeath consists of three major pathways the intrinsic pathwaythe extrinsic or death-receptor pathway and the signallingpathways that indicates death-associated proteolytic or nucle-olytic activities

The Bcl-2 family of proteins play an essential role in regu-lating the mitochondria-dependent pathway of apoptosis forhealth and diseases [19] Bcl-2 proteins are alternative targetsfor both pro- and antiapoptotic therapeutic approaches [20]There are two groups of proteins involved in apoptosismdashantiapoptotic or prosurvival proteins such as Bcl-2 Bcl-xlBcl-w Mcl-1 and A1 and proapoptotic proteins such as BaxBak PUMA Bok Bad Bid Bik Blk Hrk BNIP3 and BimLThe Bcl-2 family of proteins have been shown to regulateapoptosis in response to chemotherapy both in vivo and invitro [21 22] The Bcl-2 family of proteins are located inthe endoplasmic reticulummembrane the nuclear envelopeand in the mitochondrionrsquos outer membranes [23] The Bcl-2prosurvival proteins directly or indirectly regulate the releaseof cytochrome c from mitochondria

A member of the BH3 subgroup of the Bcl-2 family ofproteins is PUMA (p53-upregulated mediator of apoptosis)which interacts with antiapoptotic Bcl-2 family proteinssuch as Bcl-xL Bcl-2 Mcl-1 Bcl-w and A1 or proapoptoticproteins such as Bax and Bak It also involves p53-dependentand p53-independent apoptosis induced by different sig-nalling pathways Another antiapoptotic protein from theBcl-2 family is the myeloid cell leukemia sequence 1 (Mcl-1) which is involved in the upregulation of apoptosis andsurvival regulation and also plays a part in melanoma cellresistance towards Fas-mediated apoptosis Inhibiting themitochondrial signalling pathway of apoptosis can directlyaffect cancer therapy It is well known that Bcl-2 is a potentantiapoptotic protein which is expressed in HT-29 cellsIn our study the expression of apoptosis and cell cycleassociated proteins was determined using western blottingafter the HT-29 cells were treated with 2410158406-trihydroxy-4-methoxybenzophenone The expression of antiapoptotic(Bcl-2 and Mcl-1) and proapoptotic protein (PUMA andBak) in HT-29 colon human adenocarcinoma cells line wasexamined using the western blot technique To clarify themechanisms by which the compound induced apoptosis ofHT-29 cells the cells were incubated at an IC

50concentra-

tion (115 120583M) of 410158406-trihydroxy-4-methoxybenzophenonefor 24 h 48 h and 72 h Proteins were then extracted forwestern blotting As is shown in Figures 6(c) and 6(d) theexpression of Mcl-1 and PUMAwas significantly upregulatedwhen the HT-29 cells were treated with the compound for24 h 48 h and 72 hThe expression of Bcl-2 and Bak was only

BioMed Research International 9

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m Io

dide

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

Annexin V-FITC

1549

6926

1228

296

3040 2599

3516 846

1963

2283

5081

671

24h96120583M 192120583M 288120583M

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m Io

dide

185

9228

321

266

Control

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m Io

dide

Annexin V-FITC100 101 102 103 104

Annexin V-FITC

100 101 102 103 104

Prop

idiu

m Io

dide

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

Annexin V-FITC

100 101 102 103 104

Annexin V-FITC

96120583M 192120583M 288120583M

96120583M 192120583M 288120583M

48h

72h

804 3148

1961 1087

3505 2599

3422 474

1002 5467

2680 851

3069 1577

5221 133

1396 5897

1273 1434

3663 845

5464 028

Annexin V-FITC

(a)

0102030405060708090

100110

URUL

LRLL

Unt

reat

ed 96

192

288

96

192

288

96

192

288

Concentration (120583gmL)

Cel

l pop

ulat

ion

()

48h 72h24h

(b)

Figure 5 (a) Effects of 2410158406-trihydroxy-4-methoxybenzophenone on induction of apoptosis in HT-29 cells The cells were treated withdifferent concentrations of compound (25 120583M 50 120583M and 75 120583M) in a time-dependent manner (24 h 48 h and 72 h) labelled with FITCannexinV andPIViable cells = LL Early apoptotic cells = LR late or secondary necrotic cells =UR primary necrotic cells =UL (b)Histogramrepresentation of the quantitative percentage of viable cells (LL) early apoptotic cells (LR) primary necrotic cells (UL) and late apoptoticcells or secondary necrotic cells (UR) of HT-29 treatment with different concentration of 2410158406-trihydroxy-4-methoxybenzophenone for24 h 48 h and 72 h

10 BioMed Research International

2500

2000

1500

1000

500

0

0 200 400 600 800 1000

Num

ber

0 200 400 600 800 1000

1000

3500

3000

2500

2000

1500

1000

500

0

Num

ber

0 200 400 600 800

3500

3000

2500

2000

1500

1000

500

0

Num

ber

FL2-A FL2-area (DNA content)

0 200 400 600 800

4000

3000

2000

1000

0

Num

ber

FL2-A FL2-area (DNA content)

FL2-A FL2-area (DNA content) FL2-A FL2-area (DNA content)

48h 72h

24hControlG1 5600G2 1781S 2619

G1 7069G2 1361S 1570

G1 8821G2 320S 860

G1 7998G2 317S 1685

(a)

0

20

40

60

80

100

S

48h 72h24hUntreated

HT-29

Dip

loid

cells

() 90

70

50

30

10

G1G0

G2M

(b)

48h 72h24hUntreated

(c)

Mcl-1

PUMA

Bcl-2

Bak

48h 72h24hUntreated

(d)

Figure 6 (a) Effect of 2410158406-trihydroxy-4-methoxybenzophenone on HT-29 cell cycle Cells were treated with the IC50

value (30 120583M)concentrations of compound for 24 h 48 h and 72 h and analyzed by flow cytometry after staining with PI Percentages of diploid cells (DNAcontent) at G

0G1 S and G

2M phases of HT-29 cells were determined after 24 h 48 h and 72 h incubation periods (b) Histogram showing

quantitative percentage of diploid cells (DNA content) in each cell cycle phase without treatment and with treatment (c) The expression ofthe whole protein in Coomassie blue staining after compound induced apoptosis in HT-29 cells (d) The expression of antiapoptotic proteinand proapoptotic protein in western blotting analysis

up-regulated for 72 h treatment of HT-29 cells In apoptosis-related protein expression after treatmentwith the compoundin HT-29 cells the upregulation of Bcl-2 was found tobe in a time-dependent manner Our results showed thatthe G

0G1arrest and apoptosis of the 2410158406-trihydroxy-4-

methoxybenzophenone in HT-29 cells might be associatedwith the upregulation of the anti-apopptotic proteins Bcl-2 and Mcl-1 as well as the pro-apototic proteins Bak and

PUMA Recent researchers also demonstrated that DLBS1425showed downregulation of Bax and Bcl-2 which are COX-2 cPLA2 and VEGF-C and c-fos and HER-2neu mRNAexpression in MDA-MB231 cells [8] Using western blottingwe showed that Bcl-2 Mcl-1 and Bak and PUMA regulationmay be involved in compound-induced apoptosis Thus wehave shown that the Bcl-2 family of proteins may be directlyor indirectly involved in the apoptotic effect of the compound

10 BioMed Research International

2500

2000

1500

1000

500

0

0 200 400 600 800 1000

Num

ber

0 200 400 600 800 1000

1000

3500

3000

2500

2000

1500

1000

500

0

Num

ber

0 200 400 600 800

3500

3000

2500

2000

1500

1000

500

0

Num

ber

FL2-A FL2-area (DNA content)

0 200 400 600 800

4000

3000

2000

1000

0

Num

ber

FL2-A FL2-area (DNA content)

FL2-A FL2-area (DNA content) FL2-A FL2-area (DNA content)

48h 72h

24hControlG1 5600G2 1781S 2619

G1 7069G2 1361S 1570

G1 8821G2 320S 860

G1 7998G2 317S 1685

(a)

0

20

40

60

80

100

S

48h 72h24hUntreated

HT-29

Dip

loid

cells

() 90

70

50

30

10

G1G0

G2M

(b)

48h 72h24hUntreated

(c)

Mcl-1

PUMA

Bcl-2

Bak

48h 72h24hUntreated

(d)

Figure 6 (a) Effect of 2410158406-trihydroxy-4-methoxybenzophenone on HT-29 cell cycle Cells were treated with the IC50

value (30 120583M)concentrations of compound for 24 h 48 h and 72 h and analyzed by flow cytometry after staining with PI Percentages of diploid cells (DNAcontent) at G

0G1 S and G

2M phases of HT-29 cells were determined after 24 h 48 h and 72 h incubation periods (b) Histogram showing

quantitative percentage of diploid cells (DNA content) in each cell cycle phase without treatment and with treatment (c) The expression ofthe whole protein in Coomassie blue staining after compound induced apoptosis in HT-29 cells (d) The expression of antiapoptotic proteinand proapoptotic protein in western blotting analysis

up-regulated for 72 h treatment of HT-29 cells In apoptosis-related protein expression after treatmentwith the compoundin HT-29 cells the upregulation of Bcl-2 was found tobe in a time-dependent manner Our results showed thatthe G

0G1arrest and apoptosis of the 2410158406-trihydroxy-4-

methoxybenzophenone in HT-29 cells might be associatedwith the upregulation of the anti-apopptotic proteins Bcl-2 and Mcl-1 as well as the pro-apototic proteins Bak and

PUMA Recent researchers also demonstrated that DLBS1425showed downregulation of Bax and Bcl-2 which are COX-2 cPLA2 and VEGF-C and c-fos and HER-2neu mRNAexpression in MDA-MB231 cells [8] Using western blottingwe showed that Bcl-2 Mcl-1 and Bak and PUMA regulationmay be involved in compound-induced apoptosis Thus wehave shown that the Bcl-2 family of proteins may be directlyor indirectly involved in the apoptotic effect of the compound

BioMed Research International 11

in HT-29 colon human carcinoma cell lines and thatthe 2410158406-trihydroxy-4-methoxybenzophenone may induceapoptosis in HT-29 cells through pathways involving Bcl-2

4 Conclusion

Our study showed that 2410158406-trihydroxy-4-methoxyben-zophenone inhibits cell proliferation and is able to induceapoptosis in HT-29 cells Western blot analysis suggestedthat the compound induced apoptosis by upregulating Bcl-2 PUMA Mcl-1 and Bak proteins These data suggested thatthe mechanism of cell cycle arrest in the G

0G1phase may be

related to Bcl-2 proteins and play a vital role in regulating themitochondria-dependent pathway of apoptosis

Abbreviations

g GrammL Millilitermin Minutesh Hoursmz Molecular weightAOPI Acridine orange and propidium iodide

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This project was supported by grants from the Ministry ofHigher Education (nos PS2862009C and PS4702010B)Theauthors would like to acknowledge the laboratory staff ofthe Institute of Medical Research (IMR Malaysia) for theirvaluable technical assistance in the flow cytometry analysisThe authors are also thankful to Professor Dr Amru BinNasrulhaq Boyce for allowing them to use his laboratorySpecial thanks are also due to theMerit Islamic DevelopmentBank scholarship (IDB Saudi Arabia) for financial supportfor Ma Ma Lay

References

[1] L J Kleinsmith Principles of Cancer Biology PearsonBenjaminCummings 2003

[2] G M Cooper The Cancer Book Jones and Barlett BostonMass USA 1993

[3] D Kurnia K Akiyama and H Hayashi ldquo29-norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[4] E Susanthy ldquoN-hexane extract cytotoxicity Beans andMeatMahkota Dewa Fruit (Phaleria macrocarpa [Scheff] Boerl)Against HeLa and Myeloma Cell Linerdquo pp 47ndash50 2005

[5] R Hendra S Ahmad E Oskoueian A Sukari and M YShukor ldquoAntioxidant Anti-inflammatory and cytotoxicity ofPhaleria macrocarpa (Boerl) Scheff Fruitrdquo BMC Complemen-tary and Alternative Medicine vol 11 article 110 2011

[6] A Faried D Kurnia L S Faried et al ldquoAnticancer effects ofgallic acid isolated from Indonesian herbal medicine Phaleriamacrocarpa (Scheff) Boerl on human cancer cell linesrdquo Inter-national Journal of Oncology vol 30 no 3 pp 605ndash613 2007

[7] O M Tandrasasmita J S Lee S H Baek and R R Tjandraw-inata ldquoInduction of cellular apoptosis in human breast cancerby DLBS1425 a Phaleria macrocarpa compound extract viadownregulation of PI3-kinaseAKT pathwayrdquoCancer Biology ampTherapy vol 10 no 8 pp 814ndash824 2010

[8] R R Tjandrawinata P F Arifin O M Tandrasasmita DRahmi and A Aripin ldquoDLBS1425 a Phaleria macrocarpa(Scheff) Boerl extract confers anti proliferative and proapop-tosis effects via eicosanoid pathwayrdquo Journal of ExperimentalTherapeutics and Oncology vol 8 no 3 pp 187ndash201 2010

[9] A Aripin P F Arifin and R R Thandrawinata ldquoIsolatedcompounds from Phaleria macrocarpa as anti-cancer agentsrdquoUS Patent Application No 20110257112 2011

[10] Susilawati S Matsjeh H D Pranowo and C Anwar ldquoAntiox-idant activity of 2641015840-trihyroxy-4-methoxy benzophenonefrom ethyl acetate extract of leaves of Mahkota Dewa (Phaleriamacrocarpa (Scheff) Boerlrdquo Indonesian Journal of Chemistryvol 11 no 2 pp 180ndash185 2011

[11] S N A Malek C W Phang H Ibrahim N A Wahab and KS Sim ldquoPhytochemical and cytotoxic investigations of Alpiniamutica rhizomesrdquoMolecules vol 16 no 1 pp 583ndash589 2011

[12] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[13] S N A Malek S K Shin N A Wahab and H Yaacob ldquoCyto-toxic components of Pereskia bleo (Kunth) DC (Cactaceae)leavesrdquoMolecules vol 14 no 5 pp 1713ndash1724 2009

[14] S Diaz-Moralli M Tarrado-Castellarnau A Miranda and MCascante ldquoTargeting cell cycle regulation in cancer therapyrdquoPharmacology ampTherapeutics vol 138 no 2 pp 255ndash271 2013

[15] J A Call S G Eckhardt and D R Camidge ldquoTargetedmanipulation of apoptosis in cancer treatmentrdquo The LancetOncology vol 9 no 10 pp 1002ndash1011 2008

[16] M A Dickson and G K Schwartz ldquoDevelopment of cell-cycleinhibitors for cancer therapyrdquo Current Oncology vol 16 no 2pp 36ndash43 2009

[17] Y-L Cheng W-L Chang S-C Lee et al ldquoAcetone extract ofAngelica sinensis inhibits proliferation of human cancer cellsvia inducing cell cycle arrest and apoptosisrdquo Life Sciences vol75 no 13 pp 1579ndash1594 2004

[18] Z Yuan C Long T Junming L Qihuan Z Youshun and ZChan ldquoQuercetin-induced apoptosis ofHL-60 cells by reducingPI3KAktrdquo Molecular Biology Reports vol 39 no 7 pp 7785ndash7793 2012

[19] M A Masud Effect of Bioflavonoids Acacetin and Luteolin onHO-1 Human Melanoma Cancer Cells The Brooklyn CenterLong Island University 2009

[20] J C Reed and M Pellecchia ldquoApoptosis-based therapies forhematologic malignanciesrdquo Blood vol 106 no 2 pp 408ndash4182005

[21] X Qiang Investigation of programmed cell death mechanisms inArabidopsis roots during colonization with Piriformospora indica[dissertation] 2011

[22] E L Arriola A M Rodriguez-Lopez J A Hickman and C MChresta ldquoBcl-2 overexpression results in reciprocal downreg-ulation of Bcl-X(L) and sensitizes human testicular germ cell

12 BioMed Research International

tumours to chemotherapy-induced apoptosisrdquo Oncogene vol18 no 7 pp 1457ndash1464 1999

[23] F W Georg ldquoCell division and survivalrdquo in Molecular Mecha-nisms of Cancer pp 45ndash191 Springer 2007

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ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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Volume 2013

ToxinsJournal of

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ISRN Pain

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Volume 2013

ISRN Medicinal Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Advances in Pharmacological Sciences

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Pharmaceutics

ISRN Pharmaceutics

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Autoimmune Diseases

ISRN Pharmacology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

AnesthesiologyResearch and Practice

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Toxicology

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

ToxinsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Pain

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Volume 2013

ISRN Medicinal Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Advances in Pharmacological Sciences

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Pharmaceutics

ISRN Pharmaceutics

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Autoimmune Diseases

ISRN Pharmacology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

AnesthesiologyResearch and Practice

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Toxicology

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal