)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/omcl/2015/135691.pdf · each...

Research ArticleHigh Potency of a Novel Resveratrol Derivative331015840441015840-Tetrahydroxy-trans-stilbene against Ovarian Cancer IsAssociated with an Oxidative Stress-MediatedImbalance between DNA Damage Accumulation and Repair

Justyna MikuBa-Pietrasik1 Patrycja SosiNska1 Marek Murias2

Marcin Wierzchowski3 Marta BrewiNska-Olchowik4 Katarzyna Piwocka4

Dariusz Szpurek5 and Krzysztof Ksidhek1

1Department of Pathophysiology Poznan University of Medical Sciences Rokietnicka 8 Street 60-806 Poznan Poland2Department of Toxicology Poznan University of Medical Sciences Dojazd 30 Street 60-631 Poznan Poland3Department of Chemical Technology of Drugs Poznan University of Medical Sciences Grunwaldzka 6 60-780 Poznan Poland4Laboratory of Cytometry Nencki Institute of Experimental Biology Polish Academy of Sciences Pasteura 3 02-093 Warsaw Poland5Division of Gynecological Surgery Poznan University of Medical Sciences Polna 33 Street 60-535 Poznan Poland

Correspondence should be addressed to Krzysztof Ksiązek kksiazekumpedupl

Received 8 April 2015 Revised 7 June 2015 Accepted 14 June 2015

Academic Editor Gabriele Saretzki

Copyright copy 2015 Justyna Mikuła-Pietrasik et alThis is an open access article distributed under theCreativeCommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

We explored the effect of a new resveratrol (RVT) derivative 331015840441015840-tetrahydroxy-trans-stilbene (331015840441015840-THS) on viabilityapoptosis proliferation and senescence of three representative lines of ovarian cancer cells that is A2780 OVCAR-3 and SKOV-3 in vitro In addition themechanistic aspects of 331015840441015840-THS activity including cell redox homeostasis (the production of reactiveoxygen species activity of enzymatic antioxidants and magnitude of DNA damage accumulation and repair) and the activity ofcaspases (3 8 and 9) and p38 MAPK were examined The study showed that 331015840441015840-THS affects cancer cell viability much moreefficiently than its parent drug This effect coincided with increased generation of reactive oxygen species downregulated activityof superoxide dismutase and catalase and excessive accumulation of 8-hydroxy-21015840-deoxyguanosine and its insufficient repair dueto decreased expression of DNA glycosylase I Cytotoxicity elicited by 331015840441015840-THS was related to increased incidence of apoptosiswhich was mediated by caspases 3 and 9 Moreover 331015840441015840-THS inhibited cancer cell proliferation and accelerated senescencewhich was accompanied by the activation of p38 MAPK Collectively our findings indicate that 331015840441015840-THS may constitute avaluable tool in the fight against ovarian malignancy and that the anticancer capabilities of this stilbene proceed in an oxidativestress-dependent mechanism

1 Introduction

Over the past two decades resveratrol (3410158405-trihydroxy-trans-stilbene RVT) a phytoalexin that is present in grapesand red wine has generated a great deal of excitement dueto its explicit chemopreventive capabilities towards the initia-tion promotion and progression of numerous malignanciesincluding ovarian cancer [1] Studies have shown that RVTrestricts ovarian cancer cell proliferation [2] and adhesion

[3] induces apoptosis [4] modulates pathways involved inangiogenesis [5] and drug resistance [6] and sensitizes themto other chemotherapeutics [7] Low bioavailability and fastdegradation jeopardize however routine usage of RVT inpractical medicine [8]

Due to these deficiencies of RVT a number of its deriva-tives have been synthesized to develop a compound whichwill display improved pharmacokinetics andor strengthenedactivity as compared to its natural prototype Contemporary

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 135691 15 pageshttpdxdoiorg1011552015135691

2 Oxidative Medicine and Cellular Longevity

HO

OH

OH

OH

OH

HO

OH

349984005-Trihydroxy-trans-stilbene(resveratrol RVT)

3399840044998400-Tetrahydroxy-trans-stilbene(3399840044998400-THS)

Figure 1 Chemical structure of resveratrol and 331015840441015840-tetrahydroxy-trans-stilbeneThe hydroxyl group (ndashOH) in the highly reactive orthoposition is marked in the circle

research on synthetic stilbenes stems from a paradigm thatthe pharmacological and biological (eg prooxidative andantiproliferative potential) properties of stilbenes are tightlyassociated with a specific configuration of hydroxyl groupsin their aromatic rings [9 10] An analysis of the structuredependency of RVT derivative activity revealed that theintroduction of extra hydroxyl groups markedly improvedtheir biological capabilities especially anticancer activity [11]Moreover analogues characterized by a pyrogallol or catecholstructure have been found to display greater cytotoxicityagainst cancer cells than RVT itself [10] Last but not leastanalogues with two 441015840-hydroxyl groups on the stilbenebackbone have been recognized to outmatchRVTwith regardto inhibition of neoplastic conversion and to a reduction inthe proliferative and invasive capabilities of cancer cells [12]

One of these newly synthesized RVT analogues is331015840441015840-tetrahydroxy-trans-stilbene (331015840441015840-THS) whichhas recently been reported to be effective against T-cellleukemia Jurkat cells [9] Moreover the beneficial activity ofthis stilbene has also been observed with regard to the rate ofsenescence of human peritoneal mesothelial cells (HPMCs)[13] which is worth noting in view of the well-establishedcancer-promoting activity of senescent HPMCs [14 15]

Following these findings we aimed to examine the effectof 331015840441015840-THS on viability apoptosis and proliferationof ovarian cancer cells A2780 OVCAR-3 and SKOV-3in vitro In addition in order to examine the activity of331015840441015840-THS from the perspective of normal cells associatedwith the development of primary and metastatic ovariantumors experiments were also performed using normalhuman ovarian surface epithelial (HOSE) cells and primaryomental HPMCs The incidence of apoptosis was confrontedwith the activity of both the initiator (8 and 9) and theeffector (3) caspases Phenomena which might underliechanges in cell apoptosis and proliferation such as oxidativestress and cellular senescence have also been addressed Inthe context of oxidative stress special attention was paidto the production of reactive oxygen species (ROS) theactivity of the enzymatic antioxidants superoxide dismutase(SOD) and catalase (CAT) the accumulation of oxidativeDNA damage (8-hydroxy-21015840-deoxyguanosine (8-OH-dG))

and the efficiency of 8-OH-dG repair including the activityof DNA glycosylase I (hOgg1) In addition the activity of p38mitogen-activated protein kinase (MAPK) was examined Inorder to delineate the biological strength of 331015840441015840-THS acomparison was made of effects exerted by 331015840441015840-THS andits parent drug

2 Materials and Methods

21 Chemicals Unless otherwise stated all reagents werepurchased from Sigma-Aldrich Corp (St Louis MO USA)The tissue culture plastics were from Nunc (RoskildeDenmark) RVT was obtained from Sigma-AldrichCorp whereas 331015840441015840-THS (Figure 1) was synthesizedas described previously [16] Stock solutions of the stilbeneswere prepared in dimethyl sulfoxide (DMSO) and diluted ina cell culture medium to a desired final concentration

22 Cell Cultures Experiments were performed on A2780OVCAR-3 and SKOV-3 cells which are three most exten-sively used models of ovarian cancer [17] A2780 and SKOV-3 cells were obtained from ECCC (Porton Down UK) andpropagated in McCoyrsquos 5a and RPMI 1640 media respec-tively both supplemented with L-glutamine (2mmolL)penicillin (100UmL) streptomycin (100 gmL) and 10fetal bovine serum (FBS) Ovarian cancer cell line OVCAR-3 was purchased from ATCC (Rockville MD) and main-tained in RPMI 1640 medium with L-glutamine (2mmolL)HEPES (10mmolL) sodium pyruvate (1mmolL) glucose(4500mgL) and 20 FBSThe experiments were performedwith cells maintained in culture less than a month to preventtheir alienation from original cancer cell attributes

Human peritoneal mesothelial cells (HPMCs) were iso-lated from pieces of the omentum by enzymatic digestionas described in detail by Pronk et al [18] The tissues wereobtained from 8 patients undergoing elective abdominalsurgery The reasons for the surgery included an aorticaneurysm (6) and bowel obstruction (2) All cultures wereestablished from individuals with no evidence of peritonealmalignancy The age of the donors ranged from 40 to 42

Oxidative Medicine and Cellular Longevity 3

years old The cells were propagated in medium M199 sup-plemented with L-glutamine (2mM) penicillin (100UmL)streptomycin (100 120583gmL) hydrocortisone (04 120583gmL) and10 FBS Cells from the 1st-2nd passage were used in theexperiments The study on HPMCs was approved by aninstitutional bioethics committee

Normal human ovarian surface epithelial (HOSE) cellswere purchased fromScienCell Research Laboratories (Carls-bad CA USA) and maintained in Ovarian Epithelial CellMedium (OEpiCM)with growth supplements penicillin andstreptomycin The experiments were performed with HOSEcells from the 1st-2nd passage

23 Measurement of Cell Viability Cell viability was assessedwith the MTT test and with lactate dehydrogenase (LDH)release assay As per the MTT method normal and cancercells were seeded into 96-well plates at high density andallowed to attach overnight Then they were incubated ina medium containing 125mgmL of the MTT salt [3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide] for24 h at 37∘C The generated formazan was solubilized with20 sodium dodecyl sulphate and 50 NN-dimethyl-formamide Absorbance of the converted dye was recordedat 595 nm with a reference wavelength of 690 nm

As per LDH release the measurements were performedusing the commercially available Pierce LDH CytotoxicityAssay Kit (Pierce Biotechnology Rockford IL USA) In thisassay cells with damaged plasma membranes release LDHinto the culture environment then the enzyme catalyses theconversion of lactate to pyruvate which is accompanied by areduction of NAD+ to NADH Afterwards diaphorase usesNADH to reduce a tetrazolium salt to a formazan whoselevel is proportional to LDH activityThe absorbance emittedby the formazan was monitored at 490 nm with a referencewavelength of 680 nm

24 Detection of Apoptosis Identification of apoptosis wasbased on the presence of DNA condensation in cells stainedwith a fluorescent dye Hoechst 33258 (Molecular ProbesEugene OR) Cells were grown on Lab-Tek Chamber Slides(Nunc Roskilde Denmark) fixed with 4 formaldehydefor 10min and washed twice with PBS Then the cells wereincubated with Hoechst 33258 (01120583gmL) for 5min in thedark After washing with PBS stained nuclei were observedand counted under a Zeiss Axio Observer D1 fluorescencemicroscope (Carl Zeiss Jena Germany) The activity ofcaspases 3 8 and 9 was quantified in the cell lysates usingspecific colorimetric kits (AbcamCambridgeUK) accordingto the manufacturerrsquos instructions

25 Measurements of Cell Proliferation Cell proliferativepotential was determined using Cell Proliferation Kit I(PromoKine Heidelberg Germany) according to the man-ufacturerrsquos instructions In brief cells were probed for 15minwith 5-(and 6)-carboxyfluorescein diacetate a succinimidylester (CFDA SE 2 120583M) which diffuses into cells covalentlybinds to proteins and becomes fluorescent upon its hydrol-ysis by intracellular esterases The fluorescence emitted by

the cells (the dye is inherited by the daughter cells throughsuccessive divisions) was recorded using aVictor2 spectroflu-orometer (Perkin-Elmer Turku Finland) with excitation at435 nm and emission at 535 nm

26 Measurements of Reactive Oxygen Species (ROS) andEnzymatic Antioxidants ROS production was assessed incells probed with a fluorescent dye 2101584071015840-dichlorodihydro-fluorescein diacetate (H

2DCFDA 5 120583M) (Molecular Probes

Eugene USA) for 45min at 37∘C Fluorescence intensity incell lysates was monitored in a Victor2 spectrofluorometerwith excitation at 485 nm and emission at 535 nm

Total activity of superoxide dismutase (SOD) was mea-sured in cell lysates with a commercially available kit (RampDSystems Europe Abingdon UK) while the activity of cata-lase (CAT) was estimated by using the OxiSelect CatalaseActivity Assay Kit (Cell Biolabs Inc San Diego CA USA)Both assays were performed according to the manufacturerrsquosinstructions

27 Determination of DNA Damage and Repair OxidativeDNA damage was assessed by measuring the 8-OH-dGconcentration with a competitive immunoassay (Cell BiolabsInc) as per manufacturerrsquos instructions The efficiency of 8-OH-dG repair was estimated after cell recovery for 24 h instandard growth medium In order to examine the activityof DNA glycosylase I reverse transcription PCR was per-formed RNA extraction was carried out using the RNA Bee(Tel-Test Friendswood Texas USA) and purified accordingto the manufacturerrsquos protocol One microgram of RNAwas reverse transcribed into cDNA with random hexamerprimers and cDNA was subjected to PCR The profile of thereplication cycles was denaturation at 94∘C for 50 s annealingat 58∘C for 50 s and polymerization at 72∘C for 1min Ineach reaction the expression of glyceraldehyde-3-phosphatedehydrogenase (GAPDH) served as the internal control Theprimers used for PCR were as follows hOGG1 forward51015840-CTGCCTTCTGGACAATCTTT-31015840 hOGG1 reverse 51015840-TAGCCCGCCCTGTTCTTC-31015840 designed to amplify a 551-bp region GAPDH forward 51015840-CCATGGAGAAGGCTG-GGG-31015840 and GAPDH reverse 51015840-CAAAGTTGTCATGGA-TGACC-31015840 designed to amplify a 194-bp region (total num-ber of cycles 28) The PCR products were separated on3 agarose gels stained with ethidium bromide and thenvisualized

28 Detection of Senescence-Associated 120573-Galactosidase (SA-120573-Gal) The activity of SA-120573-Gal in the cell extracts wasquantified by measuring the rate of conversion of 4-methylumbelliferyl-120573-D-galactopyranoside to 4-methylum-belliferone as described in [19] In order to visualize expres-sion of the enzyme in the cancer cell cytoplasm cytochemicalstaining for SA-120573-Gal was performed according to Dimri etal [20]

29 Determination of p38 MAPK Activity Activation of p38MAPK was examined using the Fast Activated Cell-BasedELISA (FACE) p38 kit (Active Motif) according to the


Table 1 Half-maximal inhibitory concentrations (IC50) estimatedfor RVT and 331015840441015840-THS according to the results ofMTT and LDHrelease assays

MTT assay LDH release assayRVT 331015840441015840-THS RVT 331015840441015840-THS

A2780 48 120583M 4120583M 65 120583M 6 120583MOVCAR-3 480 120583M 50120583M 525 120583M 85 120583MSKOV-3 380 120583M 8120583M 346120583M 10120583MHOSE cells 485 120583M 502 120583M 421 120583M 532 120583MHPMCs 465 120583M 496 120583M 495 120583M 498 120583M

manufacturerrsquos instructions In brief cells were fixed with 4formaldehyde and incubated with a primary antibody thatrecognizes either phosphorylated p38 or total p38 overnightat 4∘C Then the cells were washed and incubated with asecondary HRP-conjugated antibody for 1 h at room temper-ature The color reaction that developed was read using aspectrofluorometer at 450 nm wavelength

The results were expressed as a ratio of phosphorylated tototal p38 activity

210 Statistics Statistical analysis was performed usingGraphPad Prism 500 software (GraphPad Software SanDiego USA) The means were compared using the repeatedmeasures analysis of variance (ANOVA) with the Newman-Keuls test as a post hoc test When appropriate the two-wayANOVA and Wilcoxon matched pairs tests were used Theresults were expressed as means plusmn SEM Differences with a 119901value lt005 were considered to be statistically significant

3 Results

31 331015840441015840-THS Is More Cytotoxic towards Ovarian CancerCells than RVT In order to examine the effect of 331015840441015840-THS on the viability of ovarian cancer cells and to compare itsactivity with RVT subconfluent cultures of A2780 OVCAR-3 and SKOV-3 cells were subjected to a wide range of stilbeneconcentrations (0ndash500120583M) for 24 h then MTT and LDHrelease assays were performed Both methods consistentlyshowed that either RVT or 331015840441015840-THS was capable ofinhibiting cancer cell viability albeit the effect exerted by331015840441015840-THS towards all three cancer cell lines that weretested was more profound (Figure 2) Moreover the valuesobtained in the cytotoxicity assays reanalyzed using Cal-cuSyn software (Biosoft Cambridge UK) revealed that thehalf-maximal inhibitory concentrations (IC

50) estimated for

331015840441015840-THSwere significantly lower than for RVT (Table 1)The cytotoxicity tests performed with HOSE cells and

HPMCs showed that both of the tested stilbenes were muchsafer to normal cells than to the cancer cells Namely theviability of HOSE cells subjected to RVT and 331015840441015840-THSwas unchanged up to a concentration of 400 120583M whilethe viability of HPMCs was maintained up to 300 120583M TheIC50

values established for both types of normal cells wereconsiderably higher than those estimated for the cancer cells(Table 1)

Table 2 Cytotoxicity of 331015840441015840-THS against ovarian cancer cellsnormal human ovarian surface epithelial (HOSE) cells and humanperitoneal mesothelial cells (HPMCs) at working concentrations of10 50 and 100120583MThe results are expressed as the percentage of theviability of control cells that were not subjected to 331015840441015840-THSTheasterisks indicate a significant difference as compared to the controlgroup

MTT assay LDH release assay10120583M 50 120583M 100 120583M 10120583M 50 120583M 100 120583M

A2780 20 plusmn 3lowast 14 plusmn 3lowast 13 plusmn 3lowast 42 plusmn 3lowast 26 plusmn 3lowast 22 plusmn 3lowast

OVCAR-3 75 plusmn 3lowast 50 plusmn 3lowast 48 plusmn 3lowast 62 plusmn 12lowast 53 plusmn 3lowast 46 plusmn 3lowast

SKOV-3 40 plusmn 2lowast 39 plusmn 4lowast 35 plusmn 2lowast 50 plusmn 2lowast 32 plusmn 12lowast 24 plusmn 7lowast

HOSE cells 105 plusmn 2 100 plusmn 4 107 plusmn 2 101 plusmn 2 100 plusmn 4 102 plusmn 2HPMCs 108 plusmn 14 100 plusmn 4 102 plusmn 2 101 plusmn 2 100 plusmn 4 102 plusmn 2

According to the IC50

values obtained using both MTTand LDH release assays three final concentrations of thestilbenes namely 10 50 and 100 120583M were selected to beused in all subsequent experiments (24-h exposure) Thecytotoxicity of the tested compounds used at these dosesagainst ovarian cancer cells HOSE cells and HPMCs isshown in Table 2

32 331015840441015840-THS Triggers Higher Generation of ROS inCancer Cells than RVT The redox-sensitive fluorescent dyeH2DCFDAwhich detects allmajor intercellular ROS includ-

ing hydrogen peroxide superoxides and peroxynitrites wasused to determine the effect of the tested stilbenes onthe magnitude of cellular oxidative stress The experimentsshowed that RVT (at 50 and 100120583M) inhibited the productionof ROS in A2780 cells whereas 331015840441015840-THS markedlyupregulated the generation of ROS with the strongest effectobserved at 100 120583M (Figure 3(a)) With regard to OVCAR-3 and SKOV-3 cells RVT stimulated the release of ROShowever the effects elicited by 331015840441015840-THSwere remarkablystronger (Figures 3(b) and 3(c))

In order to exclude the possibility that increased ROSrelease in cells subjected to 331015840441015840-THSwas related to eventsassociated with cell death for example degradation of themitochondria the incubation time was shortened to 6 h atwhich as we had noticed in the pilot studies viability of thecells remained unchanged The results of these experimentsshowed that all three cancer cell lines that were investigatedand subjected to 331015840441015840-THS at 10 50 and 100 120583M for6 h were still characterized by an increased ROS level ascompared to the control untreated cells (except for SKOV-3 cells treated with 331015840441015840-THS at 10 120583M) Moreover themagnitude of ROS release in cells treated with 331015840441015840-THSwas significantly higher as compared to those subjected toRVT in which the level of ROS upon 6-h exposure remainedunchanged (Figures 3(a)ndash3(c))

When it comes to the effect of RVT and 331015840441015840-THS onthe generation of ROS by normal cells the natural stilbenestimulated oxidant production inHOSE cells (at 100 120583M) andinHPMCs (at 10 120583M)The synthetic stilbene reduced in turnthe ROS level in the HOSE cells (at 10 and 50120583M) and in theHPMCs at 100 120583M (Table 3)


HOSE

0255075

100125150

0 4 8 50 150 250 350 450

Cel

l via

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y (

)

SKOV-3

0255075

100125150

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l via

bilit

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)

OVCAR-3

0255075

100125150

0 4 8 50 150 250 350 450

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l via

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)

A2780

0255075

100125150

0 4 8 50 150 250 350 450C

ell v

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lity

()

A2780

0255075

100125150

0 4 8 50 150 250 350 450

OVCAR-3

0255075

100125150

0 4 8 50 150 250 350 450

SKOV-3

0255075

100125150

0 4 8 50 150 250 350 450

HOSE

0255075

100125150

0 4 8 50 150 250 350 450

HPMCs

0255075

100125150

0 4 8 50 150 250 350 450

Cel

l via

bilit

y (

)

RVT 33998400 44998400-THS

Concentration (120583M)

HPMCs

0255075

100125150

0 4 8 50 150 250 350 450

RVT 33998400 44998400-THS


(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

lowast

lowastlowast lowastlowast

lowast

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lowast

lowast lowast lowast lowast lowast lowast lowast

lowastlowastlowastlowastlowastlowast

lowast

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lowast lowastlowast lowast lowast lowast

lowast

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lowastlowastlowast

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lowast

lowast

lowast

Figure 2 Effect of RVT and 331015840441015840-THS on the viability of ovarian cancer cells normal human ovarian surface epithelial (HOSE) cellsand human peritoneal mesothelial cells (HPMCs) The cytotoxicity of the stilbenes was tested using MTT ((a)ndash(e)) and LDH release assays((f)ndash(j)) The differences between the cytotoxicity curves were compared using two-way ANOVA The values obtained using the MTT andLDH releasemethods and depicted in the figures were reanalyzed with CalcuSyn to establish the IC

50for each stilbene and cancer cell line (see

Table 1) The asterisks indicate a significant difference as compared to cells exposed to RVTThe experiments were performed in octuplicate


0

100

200

300

400

ROS

leve

l (

)

A2780

Con RVT 33 44 -THS

lowast lowast

lowast

lowastlowast

lowastlowast

lowast

101 plusmn 2

101 plusmn 3 99 plusmn 4


152 plusmn 11

(a)

0

100

200

300

400OVCAR-3

Con RVT 33 44 -THS

lowast lowast lowast

lowast

lowastlowast

lowast lowast

lowast

100 plusmn 3104 plusmn 4

99 plusmn 5

119 plusmn 3


(b)

0

100

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300

400SKOV-3

Con RVT 33 44 -THS

lowastlowastlowast

lowast

lowast

lowast

lowast

101 plusmn 3102 plusmn 4102 plusmn 3

104 plusmn 7

117 plusmn 4

162 plusmn 11

(c)

50

0

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activ

ity (

)

A2780

Con RVT 33 44 -THS

lowastlowastlowast

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(d)

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200OVCAR-3

Con RVT 33 44 -THS

lowast

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Con RVT 33 44 -THS

lowast lowast

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activ

ity (

)

A2780

Con RVT 33 44 -THS

lowast

(g)

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200OVCAR-3

Con RVT 33 44 -THS

lowast lowast lowastlowast

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Con RVT 33 44 -THS

lowast

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(i)

10120583M

100120583M50120583M

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10120583M

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100120583M50120583M

Figure 3 Effect of RVT and 331015840441015840-THS on the generation of ROS ((a)ndash(c)) and the activity of SOD ((d)ndash(f)) and CAT ((g)ndash(i)) in ovariancancer cells The magnitude of ROS release was determined using a redox-sensitive fluorescence probe H

2DCFDA (5 120583M) which was added

to the medium for 1 h The activity of the antioxidative enzymes was determined in cell lysates upon 24-h cell exposure to the stilbenes usingcommercially available kits The asterisks indicate a significant difference as compared to the control group (Con) The hashes indicate asignificant difference as compared to cells subjected to RVTThe values (expressed as a percentage of the control group) and signs of statisticalsignificance located above the bars in panel (a)ndash(c) refer to experiments in which the cancer cells were subjected to RVT and 331015840441015840-THSfor 6 h The experiments were performed in octuplicate


Table 3 Effect of RVT and 331015840441015840-THS on the biological properties of HOSE cells and HPMCs The results are expressed as a percentageof values obtained for control cells that were not subjected to RVT and 331015840441015840-THS The results derive from experiments performed inoctuplicate withHOSE cells and from studies withHPMCs from 8 different donorsThe asterisks indicate a significant difference as comparedto the control group The hashes indicate a significant difference as compared to cells exposed to an equimolar concentration of RVT

Cells HOSE cells HPMCsStilbene RVT 331015840441015840-THS RVT 331015840441015840-THSDose 10 120583M 50 120583M 100 120583M 10 120583M 50 120583M 100 120583M 10120583M 50 120583M 100120583M 10 120583M 50 120583M 100 120583MROS 103 plusmn 3 105 plusmn 4 121 plusmn 7lowast 75 plusmn 4lowast 72 plusmn 3lowast 99 plusmn 4 108 plusmn 4lowast 106 plusmn 11 105 plusmn 7 104 plusmn 4 102 plusmn 3 82 plusmn 1lowast

SOD 111 plusmn 3lowast 112 plusmn 3lowast 121 plusmn 4lowast 122 plusmn 1lowast 141 plusmn 5lowast 136 plusmn 2lowast 110 plusmn 2lowast 112 plusmn 3lowast 113 plusmn 2lowast 122 plusmn 4lowast 141 plusmn 3lowast 136 plusmn 4lowast

CAT 121 plusmn 6lowast 119 plusmn 4lowast 121 plusmn 6lowast 132 plusmn 1lowast 163 plusmn 4lowast 162 plusmn 1lowast 101 plusmn 1 103 plusmn 4 118 plusmn 3lowast 112 plusmn 2lowast 123 plusmn 4lowast 154 plusmn 1lowast

8-OH-dG 103 plusmn 5 102 plusmn 5 82 plusmn 4lowast 103 plusmn 3 76 plusmn 3lowast 71 plusmn 2lowast 103 plusmn 3 103 plusmn 2 102 plusmn 5 91 plusmn 2lowast 88 plusmn 3lowast 76 plusmn 3lowast

Apoptosis 99 plusmn 2 101 plusmn 2 102 plusmn 3 101 plusmn 1 100 plusmn 2 102 plusmn 2 101 plusmn 1 101 plusmn 1 98 plusmn 3 98 plusmn 2 102 plusmn 2 100 plusmn 4Proliferation 103 plusmn 4 102 plusmn 1 104 plusmn 2 116 plusmn 2lowast 103 plusmn 1 99 plusmn 5 99 plusmn 3 103 plusmn 4 102 plusmn 2 141 plusmn 3lowast 103 plusmn 1 102 plusmn 2SA-120573-Gal 101 plusmn 2 101 plusmn 2 100 plusmn 3 99 plusmn 2 101 plusmn 3 102 plusmn 5 100 plusmn 2 102 plusmn 4 100 plusmn 1 76 plusmn 3lowast 104 plusmn 4 99 plusmn 3p38MAPK 103 plusmn 5 107 plusmn 10 108 plusmn 11 106 plusmn 5 104 plusmn 4 100 plusmn 3 113 plusmn 16 103 plusmn 2 105 plusmn 7 82 plusmn 3lowast 81 plusmn 6lowast 104 plusmn 2

33 Activity of Enzymatic Antioxidants in Cancer Cells Treatedwith 331015840441015840-THS Is Lower than in Cells Exposed to RVT Theactivity of two major antioxidative enzymes that is super-oxide dismutase (SOD) and catalase (CAT) was measuredin ovarian cancer cells exposed to RVT and 331015840441015840-THSin order to assess their protection against oxidative stressThe study regarding SOD revealed that A2780 and OVCAR-3cells subjected to RVT displayed upregulated activity of theenzyme In contrast cells exposed to 331015840441015840-THS increasedSOD at 10 120583M and decreased the activity of the enzymeat 50 and 100 120583M (Figures 3(d) and 3(e)) In the SKOV-3cells RVT failed to affect SOD activity while 331015840441015840-THSdownregulated it at 50 and 100 120583M (Figure 3(f)) As per CATactivity RVT stimulated the enzyme at 10 120583M in theOVCAR-3 cells at 50120583M in the A2780 OVCAR-3 and SKOV-3 cellsand at 100 120583M in the OVCAR-3 and SKOV-3 cells 331015840441015840-THS in turn did not change CAT activity in the A2780 cellsupregulated it in the OVCAR-3 cells at 10 120583M reduced it at 50and 100 120583M and downregulated it in the SKOV-3 cells at 50and 100 120583M (Figures 3(g)ndash3(i))

HOSE cells subjected to RVT displayed increased activityof SOD and CAT at 10 50 and 100 120583M In the case of theHPMCs RVT exerted similar stimulatory activity towardsSOD whereas with respect to CAT it upregulated its activitysolely at 100 120583M 331015840441015840-THS induced SOD and CATactivity either in the HOSE cells or in the HPMCs at 1050 and 100 120583M The effects exerted by this stilbene wereconsistently more profound as compared to those of RVT ateach concentration used (Table 3)

34 DNADamage inCells Treatedwith 331015840441015840-THS IsHigherthan in Those Exposed to RVT The concentration of 8-OH-dGwas quantified to delineate the effect of RVT and 331015840441015840-THS on the magnitude of oxidative DNA damage in ovariancancer cells It was found that Res induced DNA injury inA2780 and SKOV-3 at 50 and 100 120583M and in the OVCAR-3cells this effect was evident only at 100 120583M At the lowest doseof 10120583MRes failed to induceDNAdamage irrespective of thecancer cell line used Conversely 331015840441015840-THSwas capable ofincreasing the concentration of 8-OH-dG in the OVCAR-3

and SKOV-3 cells starting from a dose of 10 120583M whereas inthe A2780 cells only concentrations of 50 and 100 120583M wereeffective Importantly however the DNA-damaging effects of331015840441015840-THS were markedly stronger than those of Res ineach cancer cell line that was tested (Figures 4(a)ndash4(c))

The magnitude of DNA damage in HOSE cells exposedto RVT was decreased when the stilbene was used at 100 120583MIn the HPMCs RVT failed to affect the concentration of 8-OH-dG In the case of 331015840441015840-THS the level of 8-OH-dGin HOSE cells exposed to this compound was decreased at50 and 100 120583M whereas in the HPMCs the synthetic stilbenedecreased the level of 8-OH-dG at each concentration usedImportantly the effect of 331015840441015840-THS on the reduction ofDNA injury in both kinds of normal cells was considerablystronger as compared to the activity of RVT (except for theconcentration of 10120583M in the HOSE cells where the effectswere identical) (Table 3)

35 Efficiency of DNADamage Repair in Cancer Cells Exposedto 331015840441015840-THS Is Lower as Compared to Cells Exposed toRVT The cancer cells were treated with RVT and 331015840441015840-THS to cause DNA injury then they were washed and keptin standard growth medium to recover The experimentsshowed that 8-OH-dGgenerated in response to cell treatmentwith RVT returned to basal values within 24 h in all of thecell lines tested In cells treatedwith 331015840441015840-THS the level ofDNA injury upon 24-h recovery was decreased as comparedto time-point 0 (after stimulation) albeit it was still markedlyhigher than in the unstimulated cells (Figures 4(d)ndash4(f))

The results of 8-OH-dG repair efficiency were confrontedwith an analysis of the expression of mRNA for DNAglycosylase I which is a major enzyme involved in therepair of this DNA adduct The study showed that theexpression of mRNA for this enzyme was upregulated upon24-h stimulation with the stilbenes (time-point 0) but thenduring the 24-h recovery period it declined Of note in cellspreincubated with 331015840441015840-THS the mRNA expression levelwas remarkably lower as compared to values characterizingcells subjected to RVT This effect was evident in all of thecancer cell lines that were investigated (Figures 4(g)ndash4(j))


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33

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Time 0 +24

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RVT

33

998400 44

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Figure 4 Continued


GAPDH

hOgg1

hOgg1

10 1005010

(j)

10050

Time 0

RVT (120583M) 33998400 44998400-THS (120583M)

+24 hours

Figure 4 Effect of RVT and 331015840441015840-THS on the concentration of 8-OH-dG ((a)ndash(c)) efficiency of 8-OH-dG repair ((d)ndash(f)) and expressionof DNA glycosylase I (hOgg1) mRNA ((g)ndash(j)) in ovarian cancer cells The magnitude of oxidative DNA damage was assessed upon 24-h cellexposure to stilbenes using a commercially available kit The asterisks indicate a significant difference as compared to the control group(Con) The hashes indicate a significant difference as compared to cells subjected to RVT The experiments were performed in hexaplicate((a)ndash(c)) The efficiency of DNA repair ((d)ndash(f)) was evaluated in cells in which DNA injury was generated in response to 24-h incubationwith resveratrol (RVT) and 331015840441015840-THS (331015840441015840) Afterwards the cells were washed and exposed to standard growth medium for the next24 h to recover The magnitude of DNA damage was evaluated in three points before exposure to the stilbenes (minus24 h) after 24-h exposure(time 0) and after a 24-h period of recovery (+24 h) The asterisks indicate a significant difference as compared to cells not exposed tostilbenes (control group minus24 h point) The hashes indicate a significant difference as compared to cells preincubated with RVT (time-point0) The experiments were performed in hexaplicate Results shown in panel (g)ndash(i) represent the effect of resveratrol (RVT) and 331015840441015840-THS (331015840441015840) on the expression of DNA glycosylase I (hOgg1) mRNA The asterisks indicate a significant difference as compared to cellsexposed to RVTThe hashes indicate a significant difference as compared to cells preincubated with stilbenes for 24 h to generate DNA injury(time-point 0) The experiments were performed in hexaplicate A representative RT-PCR analysis of the hOgg1 mRNA level in A2780 cellssubjected to resveratrol (RVT) and 331015840441015840-THS (j)

36 Frequency of Caspase-Induced Apoptosis in Cancer CellsSubjected to 331015840441015840-THS Is Higher than in Cells Exposed toRVT Microscopic evaluation of the incidence of apoptosisin cancer cells probed with Hoechst 33258 showed that RVTinduced a moderate rise in the percentage of death cells in allof the cancer cell lines studied thus exhibiting its impact atconcentrations of 50 and 100120583M for the A2780 and SKOV-3 cells and at 100 120583M for the OVCAR-3 cells In contrastthe frequency of apoptosis in cultures treated with 331015840441015840-THS was considerably higher in each cancer cell line and theproapoptotic activity of this compound was evident startingfrom the lowest concentration of 10 120583M (Figures 5(a)ndash5(d))

When apoptotic cell death was examined in normalHOSE cells andHPMCs the experiments showed that neitherRVT nor 331015840441015840-THS influenced the frequency of this phe-nomenon irrespective of the concentration used (Table 3)

Afterwards the activity of the initiator (8 and 9) andeffector (3) caspases in ovarian cancer cells subjected to RVTand 331015840441015840-THS was examined in order to address themechanism by which both stilbenes trigger apoptosis Theexperiments showed that RVT activated caspases 9 and 3 andthis activity was evident at 50 and 100 120583M At the same timethere was no effect of RVT on the activation of caspase-8When it comes to 331015840441015840-THS it increased the activity ofthe same caspases as RVT did however the effects exertedby this analogue were much more profound in each cell linetested (Figures 5(e)ndash5(g))

37 331015840441015840-THS Inhibits Proliferation of Ovarian CancerCells More Efficiently than RVT Low-density cultures ofovarian cancer cells were subjected to 331015840441015840-THS and RVTfor 24 h and then their proliferation was examined Thestudy showed that both compounds effectively reduced theexpandability of A2780 OVCAR-3 and SKOV-3 cells and

that the effects displayed by 331015840441015840-THS were remarkablystronger as compared to RVT In all of the cancer cell typesstudied the effects exerted by 331015840441015840-THSwere evident at aslow a concentration as 10120583Mwhereas in the case of RVT sucha low dose was effective in SKOV-3 cultures only (Figures6(a)ndash6(c))

Experiments performed with normal cells revealed thatRVT used at 10 50 and 100 120583M as well as 331015840441015840-THS usedat 50 and 100120583M were unable to change the proliferativecapacity of either the HOSE cells or the HPMCs At the sametime 331015840441015840-THS used at 10 120583M significantly improved theproliferation of both types of normal cells (Table 3)

38 331015840441015840-THS Induces Premature Senescence in OvarianCancer Cells The cytoplasmic activity of SA-120573-Gal whichis widely treated as a marker of cellular senescence wasexamined in cancer cells subjected to RVT and 331015840441015840-THS The study showed that ovarian cancer cells main-tained in standard culture conditions exhibited onlymarginalactivity of the enzyme which was also reflected by a verysmall percentage of cells exhibiting its expression in classiccytochemistry Upon exposure of A2780 cells to RVT theactivity of SA-120573-Gal did not change irrespectively of theconcentration used Conversely 331015840441015840-THS increased SA-120573-Gal at each dose reaching the highest activity at as low adose as 10 120583M In the case of the OVCAR-3 and SKOV-3 cellscellular senescence was elicited either by RVT or by 331015840441015840-THS albeit the effects exerted by the synthetic stilbene weresignificantly greater than those of RVT at each concentrationthat was investigated (Figures 6(d)ndash6(g))

When the activity of SA-120573-Gal was measured in theHOSE cells both of the tested compounds failed to affectthe enzyme When it comes to HPMCs a similar effect wasobserved when RVT was used at 10 50 and 100 120583M and


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ity (

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Control 10120583M 33998400 44998400-THS 50120583M 33998400 44998400-THS 100120583M 33998400 44998400-THS

33998400 44998400-THS (120583M) 33998400 44998400-THS (120583M) 33998400 44998400-THS (120583M)RVT (120583M) RVT (120583M) RVT (120583M)

Figure 5 Effect of RVT and 331015840441015840-THS on incidence of apoptosis ((a)ndash(d)) and activity of caspases ((e)ndash(g)) in ovarian cancer cells Thepresence of apoptotic cells was evaluated according to their staining withHoechst 33258 Representative images showing the effect of 331015840441015840-THS on apoptosis in A2780 cells (d) Apoptotic cells (marked with arrows) were identified according to the presence of condensed nuclei andcharacteristic cell blebbing The activity of initiator (8 and 9) and effector (3) caspases was evaluated upon 24-h cell treatment with stilbenesusing a commercially available test The asterisks indicate a significant difference as compared to the control group The hashes indicate asignificant difference as compared to cells subjected to RVT The experiments were performed in hexaplicate

331015840441015840-THS used at 50 and 100 120583M At the same timewhen the synthetic stilbene was used at 10 120583M it significantlydecreased the activity of SA-120573-Gal (Table 3)

39 331015840441015840-THS Activates p38 MAPK More Efficiently thanRVT The p38 MAP kinase pathway (p38 MAPK) is one ofthe major signaling mechanisms that is involved in oxidative

stress-mediated apoptosis [21] and cellular senescence [22]In order to find if this is also the case with regard to apoptosisand senescence of ovarian cancer cells activation of theenzyme via phosphorylation upon treatment with RVT and331015840441015840-THSwas examinedThe study showed that inA2780cells RVT increased the activity of p38 MAPK at 100 120583Mwhile 331015840441015840-THS elicited its stimulatory activity at 50


0

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n (

)A2780

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10120583M

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SA-120573

-Gal

activ

ity (

)

Figure 6 Effect of RVT and 331015840441015840-THS on proliferative capacity ((a)ndash(c)) and activity of SA-120573-Gal ((d)ndash(g)) in ovarian cancer cells Theeffectiveness of cancer cell replication and the activity of SA-120573-Gal were measured for cells subjected to the tested compounds for 24 h usingfluorescence-based techniques Representative images show the expression of SA-120573-Gal in A2780 cells exposed to 331015840441015840-THS The arrowsindicate positive cells which were identified according to the presence of a blue precipitate within the cytoplasm (g) The asterisks indicate asignificant difference as compared to the control group (Con) The hashes indicate a significant difference as compared to cells subjected toRVT The experiments were performed in octuplicate

and 100 120583M The effects of this compound were stronger ascompared to RVT (Figure 7(a)) In OVCAR-3 and SKOV-3cells RVT failed to activate p38 MAPK while 331015840441015840-THSexerted upregulatory activity at 50 and 100 120583M (Figures 7(b)and 7(c))

Measurements of p38 MAPK activity in HOSE cells andHPMCs showed that RVT did not affect the enzyme inboth types of normal cells As per 331015840441015840-THS it failed to

change p38 MAPK activity in HOSE cells but significantlydownregulated it at 10 and 50 120583M in the HPMCs (Table 3)

4 Discussion

In this report we documented that the RVT derivative331015840441015840-THS displays remarkable activity against three rep-resentative lines of ovarian cancer cells that is A2780


0

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10120583M

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10120583M

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33998400 44998400-THS

Figure 7 Effect of RVT and 331015840441015840-THS on the activity of p38 MAPK in ovarian cancer cells The level of enzyme activity upon cancercell treatment with stilbenes for 24 h was estimated according to the ratio of phosphorylated to total p38 MAPK The asterisks indicate asignificant difference as compared to the control group The hashes indicate a significant difference as compared to cells subjected to RVTThe experiments were performed in hexaplicate

OVCAR-3 and SKOV-3 cells [17] Significantly a directcomparison of the effects exerted by 331015840441015840-THS and RVTrevealed that the synthetic stilbene acts more efficiently thanits natural precursor

The first aspect of ovarian cancer cell behavior in which331015840441015840-THS surpassed RVT was cell viability which wasaffected by this compound at much lower doses than in thecase of RVT This was especially intriguing in the case of theSKOV-3 cells which are known to be very resistant to classicchemotherapeutics [23] The higher cytotoxicity of 331015840441015840-THS as compared to RVT towards ovarian malignancy is inline with the report of Fan et al who observed that anotherRVT analogue with two hydroxyl groups in positions 4 and41015840 (441015840-dihydroxy-trans-stilbene) also dominated over itsprecursor against promyelocytic leukaemia cells [24]

Low IC50

values estimated for 331015840441015840-THS especiallyagainst A2780 and SKOV-3 cells (4ndash6120583M and 8ndash10 120583Mresp) are also very promising in the context of the sideeffects which might occur in normal cells during treatmentof primary ovarian tumor or its intraperitoneal metastasisIn fact when the toxicity of 331015840441015840-THS was tested withregard to normal human ovarian surface epithelial (HOSE)cells and human peritoneal mesothelial cells (HPMCs) thecorresponding IC

50values appeared to be several times

higher and the doses of the stilbene considered to be effectiveagainst cancer cells were entirely safe to the normal cells

It is well known that the cytotoxicity of various chemicalcompounds towards cancer cells is often underlined byhyperproduction of ROS [25] andor depletion of cellularantioxidants [26] Our studies confirmed this scenario byshowing that the generation of ROS in cancer cells exposedto 331015840441015840-THS was markedly higher as compared bothto the control group and to the cells subjected to RVTThis effect is in agreement with the study of Li et al whofound that polyhydroxylated RVT analogues exacerbate ROSrelease in cancer cells [27] At the same time cancer cellssubjected to 331015840441015840-THS were characterized by reduced

activity of the major enzymatic antioxidants SOD andCAT which resembles the findings of Murias et al whofound that another ortho polyhydroxylated RVT deriva-tive 331015840441015840551015840-hexahydroxy-trans-stilbene (331015840441015840551015840-HHS) elicited cytotoxic activity against breast cancer cellsby decreasing the expression of SOD and CAT [28] It isworth noting that cells exposed to RVT displayed increasedactivity of both antioxidative enzymes analogically to theresults of Khan et al on prostate hepatic and breast cancercells [29] which indicates that the magnitude of oxidativestress provoked in ovarian cancer cells by the natural stilbenewas significantly lower as compared to 331015840441015840-THS

Cellular toxicity driven by an imbalance in the redoxstatus (increased ROSdecreased antioxidants) is accompa-nied by an accumulation of a wide spectrum of criticalDNA modifications [30] In this project we found thateither RVT or 331015840441015840-THS induces oxidative DNA injury asevidenced by the analysis of the concentration of 8-hydroxy-21015840-deoxyguanosine (8-OH-dG) Notably the magnitude ofDNA damage in cancer cells exposed to 331015840441015840-THS wassignificantly higher Increased accumulation of DNA damagein cells treated with the synthetic RVT analogue was probablythe result of the insufficient activity of certain DNA repairmechanisms including those involved in 8-OH-dG removalIn fact the analysis of mRNA for DNA glycosylase I (hOgg1)a major enzyme involved in repairing this kind of DNAlesions [31] showed that its expression in cells recovering afterpreincubation with 331015840441015840-THS was significantly dimin-ished

A plethora of evidence suggests that oxidative stress-related cytotoxicity is associated with an induction of apop-totic cell death [32] Furthermore apoptosis is also thepredominant kind of death triggered in ovarian cancercells exposed to various chemotherapeutic agents [33 34]This was also the case for the A2780 and SKOV-3 ovar-ian cancer cells treated with the RVT analogue 34410158405-tetramethoxystilbene [35] Our current study extended this


observation since 331015840441015840-THS triggered substantial apop-tosis in all cancer cell lines tested and its activity in this regardwas considerably stronger as compared to RVT

The proapoptotic activity of 331015840441015840-THS prompted usto investigate the activation of caspases which are known tobe the primary drivers of apoptosis [36] The experimentsshowed that ovarian cancer cells treated with the syntheticstilbene exhibited upregulated activity of initiator caspase-9and effector caspase-3 At the same time there was a lack ofactivation of caspase-8 which implies that the mechanismof apoptosis elicited by 331015840441015840-THS is primarily related tothe mitochondrial pathway of programmed cell death [28]It may be speculated that activation of this apoptotic routemay be a common feature of synthetic RVT derivatives asprevious reports on 34410158405-tetramethoxystilbene [35] and331015840441015840551015840-hexahydroxy-trans-stilbene [27] revealed a sim-ilar pattern of caspase activation in cancer cellsThe inductionof mitochondria-related apoptosis in response to 331015840441015840-THSmay be to some extent coupled with declined activity ofSOD in those cells Taking into account that SOD neutralizesmitochondria-derived superoxides decreased activity of thisenzyme may sensitize mitochondrial membranes to ROS-mediated damage leading to increased release of cytochromec and apoptosis

The next aspect of ovarian cancer cell progression thatwas addressed here was the proliferative capacity The studyshowed that RVT in accordance with previous reports [237] inhibits ovarian cancer cell expandability however theantiproliferative effects produced by 331015840441015840-THS were evenstronger This observation is in agreement with a previousreport on the polyhydroxylated RVT derivative 331015840441015840551015840-HHS which inhibited the growth of colorectal cancer cells[38]This is also in line with studies on the 441015840-tetrahydroxy-trans-stilbene that suppressed the proliferation of fibroblastsand breast cancer cells more efficiently than RVT [12 39]

A reduction of cell replication may result not onlyfrom the collapse of the mitotic mechanism but also frompremature development of the senescence phenotype Thiscould also be the case for 331015840441015840-THS which induced theactivity of the senescence-indicatory enzyme SA-120573-Gal athigher efficacy than RVTThe prosenescence activity of RVTwas recently described by Luo et al who showed that thiscompound increases the expression of SA-120573-Gal in lungcancer cells and that this effect was associated with anaccumulation of oxidative DNA damage [40] An increasedlevel of generated ROS has also been recognized as theprimary reason for RVT-induced premature senescence incolon carcinoma cells [41]

As for the molecular pathways engaged in the anticanceractivity of 331015840441015840-THS we found that some role may beplayed by p38 MAPK which was found to be significantlyhyperphosphorylated in ovarian cancer cells exposed to thestilbene Activation of this enzyme in cancer cells subjectedto 331015840441015840-THS was considerably greater as compared tothose treated with RVT which may imply that similarlyto other types of cells [21 22] this pathway may act as adownstream signaling element controlling oxidative stress-mediated apoptosis and senescence in ovarian cancer cellsThis assumption is in agreement with a recent study on

colorectal cancer cells in which p38 MAPK was activated inresponse to RVT-mediated exacerbation of the ROS level [41]

Last but not least it should be mentioned that theeffects exerted by 331015840441015840-THS on ovarian cancer cells wereconfronted with those on normal HOSE cells and HPMCsThe experiments showed that in contrast to its explicitproapoptotic and antiproliferative activity against A2780OVCAR-3 and SKOV-3 cells 331015840441015840-THS did not induceapoptosis and did not impair proliferation of the normalcells Conversely this stilbene used at certain concentrationswas able to improve the expandability of both kinds ofnormal cells which was plausibly associated with its ben-eficial influence on the oxidative stress-related parametersincluding decreased production of ROS elevated activity ofenzymatic antioxidants and decreased level of DNA injuryThese effects resemble the results of previous studies onHPMCs in which 331015840441015840-THS appeared to delay theirsenescence by stimulation of antioxidative and DNA damagerepair mechanisms [42]

Taken together our study provides evidence that 331015840441015840-THS has great potential to be a valuable tool in the treatmentof ovarian cancer The mechanism by which this stilbeneexerts its action is connected with direct cytotoxicity induc-tion of apoptosis reduction of proliferative capacity andactivation of senescence Intensification of these processesseems to be governed by a set of oxidative stress-associatedevents including the excessive release of ROS diminishedcell protection by enzymatic antioxidants augmented DNAinjury and insufficient DNA repair

Conflict of Interests

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

Acknowledgment

The studywas supported by a grant from theNational ScienceCentre Poland (Decision nos DEC-201103NNZ706277and DEC-201103BNZ301214) K Piwocka is an ISACScholar Fellow 2012ndash2016

References

[1] D Delmas A Lancon D Colin B Jannin and N LatruffeldquoResveratrol as a chemopreventive agent a promising moleculefor fighting cancerrdquo Current Drug Targets vol 7 no 4 pp 423ndash442 2006

[2] N-H Kang K-A Hwang H-R Lee D-W Choi and K-C Choi ldquoResveratrol regulates the cell viability promoted by17beta-estradiol or bisphenol A via down-regulation of thecross-talk between estrogen receptor 120572 and insulin growthfactor-1 receptor in BG-1 ovarian cancer cellsrdquo Food andChemical Toxicology vol 59 pp 373ndash379 2013

[3] J Mikuła-Pietrasik P Sosinska and K Ksiązek ldquoResveratrolinhibits ovarian cancer cell adhesion to peritoneal mesotheliumin vitro by modulating the production of 12057251205731 integrins andhyaluronic acidrdquo Gynecologic Oncology vol 134 no 3 pp 624ndash630 2014


[4] C Lin D R Crawford S Lin et al ldquoInducible COX-2-depend-ent apoptosis in human ovarian cancer cellsrdquo Carcinogenesisvol 32 no 1 pp 19ndash26 2011

[5] Z Cao J Fang C Xia X Shi and B-H Jiang ldquotrans-3451015840-Trihydroxystibene inhibits hypoxia-inducible factor 1alpha andvascular endothelial growth factor expression in human ovariancancer cellsrdquo Clinical Cancer Research vol 10 no 15 pp 5253ndash5263 2004

[6] D Vergara P Simeone D Toraldo et al ldquoResveratrol down-regulates AktGSK and ERK signalling pathways in OVCAR-3 ovarian cancer cellsrdquo Molecular BioSystems vol 8 no 4 pp1078ndash1087 2012

[7] M U Nessa P Beale C Chan J Q Yu and F Huq ldquoCombina-tions of resveratrol cisplatin and oxaliplatin applied to humanovarian cancer cellsrdquo Anticancer Research vol 32 no 1 pp 53ndash59 2012

[8] TWalle F HsiehMH DeLegge J E Oatis Jr andU KWalleldquoHigh absorption but very low bioavailability of oral resveratrolin humansrdquoDrugMetabolism andDisposition vol 32 no 12 pp1377ndash1382 2004

[9] M Kucinska H Piotrowska M W Luczak et al ldquoEffectsof hydroxylated resveratrol analogs on oxidative stress andcancer cells death in human acute T cell leukemia cell lineprooxidative potential of hydroxylated resveratrol analogsrdquoChemico-Biological Interactions vol 209 no 1 pp 96ndash110 2014

[10] MMuriasW Jager N Handler et al ldquoAntioxidant prooxidantand cytotoxic activity of hydroxylated resveratrol analoguesstructure-activity relationshiprdquo Biochemical Pharmacology vol69 no 6 pp 903ndash912 2005

[11] P Saiko Z Horvath M Murias et al ldquoAntitumor effects of331015840441015840551015840-hexahydroxystilbene in HL-60 human promyelo-cytic leukemia cellsrdquoNucleosides Nucleotides and Nucleic Acidsvol 25 no 9ndash11 pp 1013ndash1017 2006

[12] M Savio T Coppa L Bianchi et al ldquoThe resveratrol analogue441015840-dihydroxy-trans-stilbene inhibits cell proliferation withhigher efficiency but differentmechanism from resveratrolrdquoTheInternational Journal of Biochemistry and Cell Biology vol 41no 12 pp 2493ndash2502 2009

[13] J Mikuła-Pietrasik P Sosinska M Murias et al ldquoResver-atrol derivative 331015840441015840-tetrahydroxy-trans-stilbene retardssenescence of mesothelial cells via hormetic-like prooxidativemechanismrdquo The Journals of Gerontology Series A BiologicalSciences and Medical Sciences 2014

[14] K Ksiazek J Mikula-Pietrasik K Korybalska G DworackiA Jorres and J Witowski ldquoSenescent peritoneal mesothelialcells promote ovarian cancer cell adhesion the role of oxidativestress-induced fibronectinrdquo American Journal of Pathology vol174 no 4 pp 1230ndash1240 2009

[15] K Ksiazek JMikuła-Pietrasik R Catar et al ldquoOxidative stress-dependent increase in ICAM-1 expression promotes adhesionof colorectal and pancreatic cancers to the senescent peritonealmesotheliumrdquo International Journal of Cancer vol 127 no 2 pp293ndash303 2010

[16] MMurias N Handler T Erker et al ldquoResveratrol analogues asselective cyclooxygenase-2 inhibitors synthesis and structure-activity relationshiprdquo Bioorganic and Medicinal Chemistry vol12 no 21 pp 5571ndash5578 2004

[17] S Domcke R Sinha D A Levine C Sander and N SchultzldquoEvaluating cell lines as tumour models by comparison ofgenomic profilesrdquo Nature Communications vol 4 article 21262013

[18] A Pronk P Leguit A A GMH van Papendrecht E HagelenT J M V van Vroonhoven andH A Verbrugh ldquoA cobblestonecell isolated from the human omentum the mesothelial cellisolation identification and growth characteristicsrdquo In VitroCellular amp Developmental BiologymdashAnimal vol 29 no 2 pp127ndash134 1993

[19] R K Gary and SM Kindell ldquoQuantitative assay of senescence-associated beta-galactosidase activity in mammalian cellextractsrdquo Analytical Biochemistry vol 343 no 2 pp 329ndash3342005

[20] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995

[21] S Zhuang J T Demirs and I E Kochevar ldquop38 mitogen-activated protein kinase mediates bid cleavage mitochondrialdysfunction and caspase-3 activation during apoptosis inducedby singlet oxygen but not by hydrogen peroxiderdquo Journal ofBiological Chemistry vol 275 no 34 pp 25939ndash25948 2000

[22] F Debacq-Chainiaux E Boilan J Dedessus Le Moutier GWeemaels and O Toussaint ldquop38(MAPK) in the senescenceof human and murine fibroblastsrdquo in Protein Metabolism andHomeostasis in Aging vol 694 of Advances in ExperimentalMedicine and Biology pp 126ndash137 Springer 2010

[23] H Wahl L Tan K Griffith M Choi and J R Liu ldquoCurcuminenhances Apo2LTRAIL-induced apoptosis in chemoresistantovarian cancer cellsrdquo Gynecologic Oncology vol 105 no 1 pp104ndash112 2007

[24] G-J Fan X-D Liu Y-P Qian et al ldquo441015840-Dihydroxy-trans-stilbene a resveratrol analogue exhibited enhanced antioxidantactivity and cytotoxicityrdquo Bioorganic and Medicinal Chemistryvol 17 no 6 pp 2360ndash2365 2009

[25] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009

[26] X Li X Lu H Xu et al ldquoPaclitaxeltetrandrine coloadednanoparticles effectively promote the apoptosis of gastric cancercells based on lsquooxidation therapyrsquordquo Molecular Pharmaceuticsvol 9 no 2 pp 222ndash229 2012

[27] H Li W K K Wu Z Zheng et al ldquo3310158404551015840-pentahydroxy-trans-stilbene a resveratrol derivative induces apoptosis in col-orectal carcinoma cells via oxidative stressrdquo European Journal ofPharmacology vol 637 no 1ndash3 pp 55ndash61 2010

[28] MMuriasMW Luczak ANiepsuj et al ldquoCytotoxic activity of331015840441015840551015840-hexahydroxystilbene against breast cancer cells ismediated by induction of p53 and downregulation ofmitochon-drial superoxide dismutaserdquo Toxicology in Vitro vol 22 no 5pp 1361ndash1370 2008

[29] M A Khan H-C Chen X-X Wan et al ldquoRegulatory effectsof resveratrol on antioxidant enzymes a mechanism of growthinhibition and apoptosis induction in cancer cellsrdquo Moleculesand Cells vol 35 no 3 pp 219ndash225 2013

[30] C Molzer B Pfleger E Putz et al ldquoIn vitro DNA-damagingeffects of intestinal and related tetrapyrroles in human cancercellsrdquo Experimental Cell Research vol 319 no 4 pp 536ndash5452013

[31] S Boiteux and J P Radicella ldquoThe human OGG1 genestructure functions and its implication in the process ofcarcinogenesisrdquo Archives of Biochemistry and Biophysics vol377 no 1 pp 1ndash8 2000


[32] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009

[33] V N Ayyagari and L Brard ldquoBithionol inhibits ovarian cancercell growth In Vitromdashstudies on mechanism(s) of actionrdquo BMCCancer vol 14 no 1 article 61 2014

[34] A Marczak B Bukowska and A Rogalska ldquoWP 631 andEpo B synergize in SKOV-3 human ovarian cancer cellsrdquoEnvironmental Toxicology and Pharmacology vol 37 no 1 pp256ndash266 2014

[35] H Piotrowska K Myszkowski A Ziołkowska et al ldquoResver-atrol analogue 34410158405-tetramethoxystilbene inhibits growtharrests cell cycle and induces apoptosis in ovarian SKOV-3 andA-2780 cancer cellsrdquo Toxicology and Applied Pharmacology vol263 no 1 pp 53ndash60 2012

[36] I Chowdhury B Tharakan and G K Bhat ldquoCaspasesmdashan updaterdquo Comparative Biochemistry and Physiology Part BBiochemistry and Molecular Biology vol 151 pp 10ndash27 2008

[37] M H G Raj Z Y Abd Elmageed J Zhou et al ldquoSynergisticaction of dietary phyto-antioxidants on survival and prolifera-tion of ovarian cancer cellsrdquo Gynecologic Oncology vol 110 no3 pp 432ndash438 2008

[38] P Saiko M Pemberger Z Horvath et al ldquoNovel resveratrolanalogs induce apoptosis and cause cell cycle arrest in HT29human colon cancer cells inhibition of ribonucleotide reduc-tase activityrdquo Oncology Reports vol 19 no 6 pp 1621ndash16262008

[39] C Maccario M Savio D Ferraro et al ldquoThe resveratrolanalog 441015840-dihydroxy-trans-stilbene suppresses transforma-tion in normal mouse fibroblasts and inhibits proliferation andinvasion of human breast cancer cellsrdquo Carcinogenesis vol 33no 11 pp 2172ndash2180 2012

[40] H Luo A Yang B A SchulteM JWargovich andG YWangldquoResveratrol induces premature senescence in lung cancer cellsvia ROS-mediated DNA damagerdquo PLoS ONE vol 8 no 3Article ID e60065 2013

[41] E H Heiss Y D C Schilder and V M Dirsch ldquoChronictreatment with resveratrol induces redox stress- and ataxiatelangiectasia-mutated (ATM)-dependent senescence in p53-positive cancer cellsrdquo The Journal of Biological Chemistry vol282 no 37 pp 26759ndash26766 2007

[42] J Miku a-Pietrasik P Sosi ska M Murias et al ldquoResver-atrol derivative 331015840441015840-tetrahydroxy-trans-stilbene retardssenescence of mesothelial cells via hormetic-like prooxidativemechanismrdquo The Journals of Gerontology Series A BiologicalSciences and Medical Sciences 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


HO

OH

OH

OH

OH

HO

OH

349984005-Trihydroxy-trans-stilbene(resveratrol RVT)

3399840044998400-Tetrahydroxy-trans-stilbene(3399840044998400-THS)

Figure 1 Chemical structure of resveratrol and 331015840441015840-tetrahydroxy-trans-stilbeneThe hydroxyl group (ndashOH) in the highly reactive orthoposition is marked in the circle

research on synthetic stilbenes stems from a paradigm thatthe pharmacological and biological (eg prooxidative andantiproliferative potential) properties of stilbenes are tightlyassociated with a specific configuration of hydroxyl groupsin their aromatic rings [9 10] An analysis of the structuredependency of RVT derivative activity revealed that theintroduction of extra hydroxyl groups markedly improvedtheir biological capabilities especially anticancer activity [11]Moreover analogues characterized by a pyrogallol or catecholstructure have been found to display greater cytotoxicityagainst cancer cells than RVT itself [10] Last but not leastanalogues with two 441015840-hydroxyl groups on the stilbenebackbone have been recognized to outmatchRVTwith regardto inhibition of neoplastic conversion and to a reduction inthe proliferative and invasive capabilities of cancer cells [12]

One of these newly synthesized RVT analogues is331015840441015840-tetrahydroxy-trans-stilbene (331015840441015840-THS) whichhas recently been reported to be effective against T-cellleukemia Jurkat cells [9] Moreover the beneficial activity ofthis stilbene has also been observed with regard to the rate ofsenescence of human peritoneal mesothelial cells (HPMCs)[13] which is worth noting in view of the well-establishedcancer-promoting activity of senescent HPMCs [14 15]

Following these findings we aimed to examine the effectof 331015840441015840-THS on viability apoptosis and proliferationof ovarian cancer cells A2780 OVCAR-3 and SKOV-3in vitro In addition in order to examine the activity of331015840441015840-THS from the perspective of normal cells associatedwith the development of primary and metastatic ovariantumors experiments were also performed using normalhuman ovarian surface epithelial (HOSE) cells and primaryomental HPMCs The incidence of apoptosis was confrontedwith the activity of both the initiator (8 and 9) and theeffector (3) caspases Phenomena which might underliechanges in cell apoptosis and proliferation such as oxidativestress and cellular senescence have also been addressed Inthe context of oxidative stress special attention was paidto the production of reactive oxygen species (ROS) theactivity of the enzymatic antioxidants superoxide dismutase(SOD) and catalase (CAT) the accumulation of oxidativeDNA damage (8-hydroxy-21015840-deoxyguanosine (8-OH-dG))

and the efficiency of 8-OH-dG repair including the activityof DNA glycosylase I (hOgg1) In addition the activity of p38mitogen-activated protein kinase (MAPK) was examined Inorder to delineate the biological strength of 331015840441015840-THS acomparison was made of effects exerted by 331015840441015840-THS andits parent drug

2 Materials and Methods

21 Chemicals Unless otherwise stated all reagents werepurchased from Sigma-Aldrich Corp (St Louis MO USA)The tissue culture plastics were from Nunc (RoskildeDenmark) RVT was obtained from Sigma-AldrichCorp whereas 331015840441015840-THS (Figure 1) was synthesizedas described previously [16] Stock solutions of the stilbeneswere prepared in dimethyl sulfoxide (DMSO) and diluted ina cell culture medium to a desired final concentration

22 Cell Cultures Experiments were performed on A2780OVCAR-3 and SKOV-3 cells which are three most exten-sively used models of ovarian cancer [17] A2780 and SKOV-3 cells were obtained from ECCC (Porton Down UK) andpropagated in McCoyrsquos 5a and RPMI 1640 media respec-tively both supplemented with L-glutamine (2mmolL)penicillin (100UmL) streptomycin (100 gmL) and 10fetal bovine serum (FBS) Ovarian cancer cell line OVCAR-3 was purchased from ATCC (Rockville MD) and main-tained in RPMI 1640 medium with L-glutamine (2mmolL)HEPES (10mmolL) sodium pyruvate (1mmolL) glucose(4500mgL) and 20 FBSThe experiments were performedwith cells maintained in culture less than a month to preventtheir alienation from original cancer cell attributes

Human peritoneal mesothelial cells (HPMCs) were iso-lated from pieces of the omentum by enzymatic digestionas described in detail by Pronk et al [18] The tissues wereobtained from 8 patients undergoing elective abdominalsurgery The reasons for the surgery included an aorticaneurysm (6) and bowel obstruction (2) All cultures wereestablished from individuals with no evidence of peritonealmalignancy The age of the donors ranged from 40 to 42























3 Results


50) estimated for

















HOSE

0255075

100125150

0 4 8 50 150 250 350 450

Cel

l via

bilit

y (

)

SKOV-3

0255075

100125150

0 4 8 50 150 250 350 450

Cel

l via

bilit

y (

)

OVCAR-3

0255075

100125150

0 4 8 50 150 250 350 450

Cel

l via

bilit

y (

)

A2780

0255075

100125150

0 4 8 50 150 250 350 450C

ell v

iabi

lity

()

A2780

0255075

100125150

0 4 8 50 150 250 350 450

OVCAR-3

0255075

100125150

0 4 8 50 150 250 350 450

SKOV-3

0255075

100125150

0 4 8 50 150 250 350 450

HOSE

0255075

100125150

0 4 8 50 150 250 350 450

HPMCs

0255075

100125150

0 4 8 50 150 250 350 450

Cel

l via

bilit

y (

)

RVT 33998400 44998400-THS


HPMCs

0255075

100125150

0 4 8 50 150 250 350 450

RVT 33998400 44998400-THS


(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

lowast


lowast

lowast

lowast



lowast


lowast

lowastlowast


lowast

lowast

lowastlowast

lowastlowastlowast

lowastlowast

lowast

lowast

lowast





0

100

200

300

400

ROS

leve

l (

)

A2780

Con RVT 33 44 -THS

lowast lowast

lowast

lowastlowast

lowastlowast

lowast

101 plusmn 2



152 plusmn 11

(a)

0

100

200

300

400OVCAR-3

Con RVT 33 44 -THS


lowast

lowastlowast

lowast lowast

lowast


99 plusmn 5

119 plusmn 3


(b)

0

100

200

300

400SKOV-3

Con RVT 33 44 -THS

lowastlowastlowast

lowast

lowast

lowast

lowast


104 plusmn 7

117 plusmn 4

162 plusmn 11

(c)

50

0

100

150

200

SOD

activ

ity (

)

A2780

Con RVT 33 44 -THS

lowastlowastlowast

lowastlowast

lowast

(d)

0

50

100

150

200OVCAR-3

Con RVT 33 44 -THS

lowast

lowast

lowastlowast

lowast

lowast

(e)

0

50

100

150

200SKOV-3

Con RVT 33 44 -THS

lowast lowast

(f)

0

50

100

150

200

CAT

activ

ity (

)

A2780

Con RVT 33 44 -THS

lowast

(g)

0

50

100

150

200OVCAR-3

Con RVT 33 44 -THS


lowastlowast

(h)

0

50

100

150

200SKOV-3

Con RVT 33 44 -THS

lowast

lowast

lowastlowast

(i)

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M



















0

50

100

150

2008-

OH

-dG

leve

l (

)A2780

lowast lowast

Con RVT 33 44 -THS

lowast lowast

(a)

0

50

100

150

200OVCAR-3

lowast

Con RVT 33 44 -THS

lowastlowastlowast

(b)

0

50

100

150

200SKOV-3

lowastlowast

Con RVT 33 44 -THS

lowastlowast

lowast

(c)

0

50

100

150

200

8-O

H-d

G le

vel (

)

RVT

A2780


lowastlowast

minus24 0 +24

Stan

dard

med

ium

(d)

0

50

100

150

200OVCAR-3

lowastlowastlowast

lowast

lowastlowast

RVT

Stan

dard

med

ium

minus24 0 +24

(e)

0

50

100

150

200SKOV-3


lowastlowast

lowast

RVT

Stan

dard

med

ium

minus24 0 +24

(f)


(g) (h) (i)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400

10 120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

0

1

2

3

4A2780

lowast lowast

Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

Time 0 +24

Stan

dard

med

ium

RVT

33

998400 44

998400

hOgg

1G

APD

H(m

RNA

OD

timesm

m2)

lowast lowast

0

1

2

3

4OVCAR-3

lowast lowast

lowast

Time 0Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

+24

Stan

dard

med

ium

RVT

33

998400 44

998400

lowastlowast

0

1

2

3

4SKOV-3

lowast lowastlowast

Time 0Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

Stan

dard

med

ium

RVT

33

998400 44

998400

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

+24

lowast lowast

Figure 4 Continued


GAPDH

hOgg1

hOgg1

10 1005010

(j)

10050

Time 0

RVT (120583M) 33998400 44998400-THS (120583M)

+24 hours











0

20

40

60

80

100

Apop

totic

cells

()

A2780

Con RVT

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100OVCAR-3

Con RVT

lowast

lowastlowastlowast

(b)

0

20

40

60

80

100SKOV-3

Con RVT

lowastlowast

lowast

lowast

lowast

(c)

(d)

0

50

100

150

200

250

Caspase-3

Caspase-9Caspase-8

Casp

ase a

ctiv

ity (

)

A2780

10050101005010C


lowast lowast

lowast

lowast

(e)

0

50

100

150

200OVCAR-3

Caspase-3

Caspase-9Caspase-8

10050101005010C


lowastlowast

lowast

lowast

(f)

0

50

100

150

200SKOV-3

Caspase-3

Caspase-9Caspase-8

10050101005010C

lowastlowast

lowastlowast

lowastlowastlowast

lowast

(g)

10120583M

100120583M50120583M

33998400 44998400-THS

10120583M

100120583M50120583M

33998400 44998400-THS

10120583M

100120583M50120583M

33998400 44998400-THS








0

25

50

75

100

125

150

Cel

l pro

lifer

atio

n (

)A2780

lowast

lowast lowastlowast

lowast

(a)

0

25

50

75

100

125

150

OVCAR-3

lowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

SKOV-3

lowastlowast

lowast

lowastlowastlowast

(c)

0

50

100

150

200

250A2780

lowastlowastlowast

(d)

0

100

200

300

400

500OVCAR-3

lowastlowast

lowast

lowast

lowast lowast

(e)

0

100

200

300

400SKOV-3

lowastlowast

lowastlowastlowast

lowast

(f)

(g)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THS


SA-120573

-Gal

activ

ity (

)





4 Discussion



0

50

100

150

200

Phos

phor

ylat

ed p

38to

tal p

38 (

) A2780

lowastlowast

lowast

(a)

0

25

50

75

100

125

150OVCAR-3

lowastlowast

(b)

0

25

50

75

100

125

150SKOV-3

lowast lowast

(c)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS




Low IC50



















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































3 Results


50) estimated for

















HOSE

0255075

100125150

0 4 8 50 150 250 350 450

Cel

l via

bilit

y (

)

SKOV-3

0255075

100125150

0 4 8 50 150 250 350 450

Cel

l via

bilit

y (

)

OVCAR-3

0255075

100125150

0 4 8 50 150 250 350 450

Cel

l via

bilit

y (

)

A2780

0255075

100125150

0 4 8 50 150 250 350 450C

ell v

iabi

lity

()

A2780

0255075

100125150

0 4 8 50 150 250 350 450

OVCAR-3

0255075

100125150

0 4 8 50 150 250 350 450

SKOV-3

0255075

100125150

0 4 8 50 150 250 350 450

HOSE

0255075

100125150

0 4 8 50 150 250 350 450

HPMCs

0255075

100125150

0 4 8 50 150 250 350 450

Cel

l via

bilit

y (

)

RVT 33998400 44998400-THS


HPMCs

0255075

100125150

0 4 8 50 150 250 350 450

RVT 33998400 44998400-THS


(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

lowast


lowast

lowast

lowast



lowast


lowast

lowastlowast


lowast

lowast

lowastlowast

lowastlowastlowast

lowastlowast

lowast

lowast

lowast





0

100

200

300

400

ROS

leve

l (

)

A2780

Con RVT 33 44 -THS

lowast lowast

lowast

lowastlowast

lowastlowast

lowast

101 plusmn 2



152 plusmn 11

(a)

0

100

200

300

400OVCAR-3

Con RVT 33 44 -THS


lowast

lowastlowast

lowast lowast

lowast


99 plusmn 5

119 plusmn 3


(b)

0

100

200

300

400SKOV-3

Con RVT 33 44 -THS

lowastlowastlowast

lowast

lowast

lowast

lowast


104 plusmn 7

117 plusmn 4

162 plusmn 11

(c)

50

0

100

150

200

SOD

activ

ity (

)

A2780

Con RVT 33 44 -THS

lowastlowastlowast

lowastlowast

lowast

(d)

0

50

100

150

200OVCAR-3

Con RVT 33 44 -THS

lowast

lowast

lowastlowast

lowast

lowast

(e)

0

50

100

150

200SKOV-3

Con RVT 33 44 -THS

lowast lowast

(f)

0

50

100

150

200

CAT

activ

ity (

)

A2780

Con RVT 33 44 -THS

lowast

(g)

0

50

100

150

200OVCAR-3

Con RVT 33 44 -THS


lowastlowast

(h)

0

50

100

150

200SKOV-3

Con RVT 33 44 -THS

lowast

lowast

lowastlowast

(i)

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M



















0

50

100

150

2008-

OH

-dG

leve

l (

)A2780

lowast lowast

Con RVT 33 44 -THS

lowast lowast

(a)

0

50

100

150

200OVCAR-3

lowast

Con RVT 33 44 -THS

lowastlowastlowast

(b)

0

50

100

150

200SKOV-3

lowastlowast

Con RVT 33 44 -THS

lowastlowast

lowast

(c)

0

50

100

150

200

8-O

H-d

G le

vel (

)

RVT

A2780


lowastlowast

minus24 0 +24

Stan

dard

med

ium

(d)

0

50

100

150

200OVCAR-3

lowastlowastlowast

lowast

lowastlowast

RVT

Stan

dard

med

ium

minus24 0 +24

(e)

0

50

100

150

200SKOV-3


lowastlowast

lowast

RVT

Stan

dard

med

ium

minus24 0 +24

(f)


(g) (h) (i)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400

10 120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

0

1

2

3

4A2780

lowast lowast

Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

Time 0 +24

Stan

dard

med

ium

RVT

33

998400 44

998400

hOgg

1G

APD

H(m

RNA

OD

timesm

m2)

lowast lowast

0

1

2

3

4OVCAR-3

lowast lowast

lowast

Time 0Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

+24

Stan

dard

med

ium

RVT

33

998400 44

998400

lowastlowast

0

1

2

3

4SKOV-3

lowast lowastlowast

Time 0Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

Stan

dard

med

ium

RVT

33

998400 44

998400

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

+24

lowast lowast

Figure 4 Continued


GAPDH

hOgg1

hOgg1

10 1005010

(j)

10050

Time 0

RVT (120583M) 33998400 44998400-THS (120583M)

+24 hours











0

20

40

60

80

100

Apop

totic

cells

()

A2780

Con RVT

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100OVCAR-3

Con RVT

lowast

lowastlowastlowast

(b)

0

20

40

60

80

100SKOV-3

Con RVT

lowastlowast

lowast

lowast

lowast

(c)

(d)

0

50

100

150

200

250

Caspase-3

Caspase-9Caspase-8

Casp

ase a

ctiv

ity (

)

A2780

10050101005010C


lowast lowast

lowast

lowast

(e)

0

50

100

150

200OVCAR-3

Caspase-3

Caspase-9Caspase-8

10050101005010C


lowastlowast

lowast

lowast

(f)

0

50

100

150

200SKOV-3

Caspase-3

Caspase-9Caspase-8

10050101005010C

lowastlowast

lowastlowast

lowastlowastlowast

lowast

(g)

10120583M

100120583M50120583M

33998400 44998400-THS

10120583M

100120583M50120583M

33998400 44998400-THS

10120583M

100120583M50120583M

33998400 44998400-THS








0

25

50

75

100

125

150

Cel

l pro

lifer

atio

n (

)A2780

lowast

lowast lowastlowast

lowast

(a)

0

25

50

75

100

125

150

OVCAR-3

lowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

SKOV-3

lowastlowast

lowast

lowastlowastlowast

(c)

0

50

100

150

200

250A2780

lowastlowastlowast

(d)

0

100

200

300

400

500OVCAR-3

lowastlowast

lowast

lowast

lowast lowast

(e)

0

100

200

300

400SKOV-3

lowastlowast

lowastlowastlowast

lowast

(f)

(g)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THS


SA-120573

-Gal

activ

ity (

)





4 Discussion



0

50

100

150

200

Phos

phor

ylat

ed p

38to

tal p

38 (

) A2780

lowastlowast

lowast

(a)

0

25

50

75

100

125

150OVCAR-3

lowastlowast

(b)

0

25

50

75

100

125

150SKOV-3

lowast lowast

(c)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS




Low IC50



















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HOSE

0255075

100125150

0 4 8 50 150 250 350 450

Cel

l via

bilit

y (

)

SKOV-3

0255075

100125150

0 4 8 50 150 250 350 450

Cel

l via

bilit

y (

)

OVCAR-3

0255075

100125150

0 4 8 50 150 250 350 450

Cel

l via

bilit

y (

)

A2780

0255075

100125150

0 4 8 50 150 250 350 450C

ell v

iabi

lity

()

A2780

0255075

100125150

0 4 8 50 150 250 350 450

OVCAR-3

0255075

100125150

0 4 8 50 150 250 350 450

SKOV-3

0255075

100125150

0 4 8 50 150 250 350 450

HOSE

0255075

100125150

0 4 8 50 150 250 350 450

HPMCs

0255075

100125150

0 4 8 50 150 250 350 450

Cel

l via

bilit

y (

)

RVT 33998400 44998400-THS


HPMCs

0255075

100125150

0 4 8 50 150 250 350 450

RVT 33998400 44998400-THS


(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

lowast


lowast

lowast

lowast



lowast


lowast

lowastlowast


lowast

lowast

lowastlowast

lowastlowastlowast

lowastlowast

lowast

lowast

lowast





0

100

200

300

400

ROS

leve

l (

)

A2780

Con RVT 33 44 -THS

lowast lowast

lowast

lowastlowast

lowastlowast

lowast

101 plusmn 2



152 plusmn 11

(a)

0

100

200

300

400OVCAR-3

Con RVT 33 44 -THS


lowast

lowastlowast

lowast lowast

lowast


99 plusmn 5

119 plusmn 3


(b)

0

100

200

300

400SKOV-3

Con RVT 33 44 -THS

lowastlowastlowast

lowast

lowast

lowast

lowast


104 plusmn 7

117 plusmn 4

162 plusmn 11

(c)

50

0

100

150

200

SOD

activ

ity (

)

A2780

Con RVT 33 44 -THS

lowastlowastlowast

lowastlowast

lowast

(d)

0

50

100

150

200OVCAR-3

Con RVT 33 44 -THS

lowast

lowast

lowastlowast

lowast

lowast

(e)

0

50

100

150

200SKOV-3

Con RVT 33 44 -THS

lowast lowast

(f)

0

50

100

150

200

CAT

activ

ity (

)

A2780

Con RVT 33 44 -THS

lowast

(g)

0

50

100

150

200OVCAR-3

Con RVT 33 44 -THS


lowastlowast

(h)

0

50

100

150

200SKOV-3

Con RVT 33 44 -THS

lowast

lowast

lowastlowast

(i)

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M



















0

50

100

150

2008-

OH

-dG

leve

l (

)A2780

lowast lowast

Con RVT 33 44 -THS

lowast lowast

(a)

0

50

100

150

200OVCAR-3

lowast

Con RVT 33 44 -THS

lowastlowastlowast

(b)

0

50

100

150

200SKOV-3

lowastlowast

Con RVT 33 44 -THS

lowastlowast

lowast

(c)

0

50

100

150

200

8-O

H-d

G le

vel (

)

RVT

A2780


lowastlowast

minus24 0 +24

Stan

dard

med

ium

(d)

0

50

100

150

200OVCAR-3

lowastlowastlowast

lowast

lowastlowast

RVT

Stan

dard

med

ium

minus24 0 +24

(e)

0

50

100

150

200SKOV-3


lowastlowast

lowast

RVT

Stan

dard

med

ium

minus24 0 +24

(f)


(g) (h) (i)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400

10 120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

0

1

2

3

4A2780

lowast lowast

Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

Time 0 +24

Stan

dard

med

ium

RVT

33

998400 44

998400

hOgg

1G

APD

H(m

RNA

OD

timesm

m2)

lowast lowast

0

1

2

3

4OVCAR-3

lowast lowast

lowast

Time 0Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

+24

Stan

dard

med

ium

RVT

33

998400 44

998400

lowastlowast

0

1

2

3

4SKOV-3

lowast lowastlowast

Time 0Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

Stan

dard

med

ium

RVT

33

998400 44

998400

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

+24

lowast lowast

Figure 4 Continued


GAPDH

hOgg1

hOgg1

10 1005010

(j)

10050

Time 0

RVT (120583M) 33998400 44998400-THS (120583M)

+24 hours











0

20

40

60

80

100

Apop

totic

cells

()

A2780

Con RVT

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100OVCAR-3

Con RVT

lowast

lowastlowastlowast

(b)

0

20

40

60

80

100SKOV-3

Con RVT

lowastlowast

lowast

lowast

lowast

(c)

(d)

0

50

100

150

200

250

Caspase-3

Caspase-9Caspase-8

Casp

ase a

ctiv

ity (

)

A2780

10050101005010C


lowast lowast

lowast

lowast

(e)

0

50

100

150

200OVCAR-3

Caspase-3

Caspase-9Caspase-8

10050101005010C


lowastlowast

lowast

lowast

(f)

0

50

100

150

200SKOV-3

Caspase-3

Caspase-9Caspase-8

10050101005010C

lowastlowast

lowastlowast

lowastlowastlowast

lowast

(g)

10120583M

100120583M50120583M

33998400 44998400-THS

10120583M

100120583M50120583M

33998400 44998400-THS

10120583M

100120583M50120583M

33998400 44998400-THS








0

25

50

75

100

125

150

Cel

l pro

lifer

atio

n (

)A2780

lowast

lowast lowastlowast

lowast

(a)

0

25

50

75

100

125

150

OVCAR-3

lowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

SKOV-3

lowastlowast

lowast

lowastlowastlowast

(c)

0

50

100

150

200

250A2780

lowastlowastlowast

(d)

0

100

200

300

400

500OVCAR-3

lowastlowast

lowast

lowast

lowast lowast

(e)

0

100

200

300

400SKOV-3

lowastlowast

lowastlowastlowast

lowast

(f)

(g)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THS


SA-120573

-Gal

activ

ity (

)





4 Discussion



0

50

100

150

200

Phos

phor

ylat

ed p

38to

tal p

38 (

) A2780

lowastlowast

lowast

(a)

0

25

50

75

100

125

150OVCAR-3

lowastlowast

(b)

0

25

50

75

100

125

150SKOV-3

lowast lowast

(c)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS




Low IC50



















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

100

200

300

400

ROS

leve

l (

)

A2780

Con RVT 33 44 -THS

lowast lowast

lowast

lowastlowast

lowastlowast

lowast

101 plusmn 2



152 plusmn 11

(a)

0

100

200

300

400OVCAR-3

Con RVT 33 44 -THS


lowast

lowastlowast

lowast lowast

lowast


99 plusmn 5

119 plusmn 3


(b)

0

100

200

300

400SKOV-3

Con RVT 33 44 -THS

lowastlowastlowast

lowast

lowast

lowast

lowast


104 plusmn 7

117 plusmn 4

162 plusmn 11

(c)

50

0

100

150

200

SOD

activ

ity (

)

A2780

Con RVT 33 44 -THS

lowastlowastlowast

lowastlowast

lowast

(d)

0

50

100

150

200OVCAR-3

Con RVT 33 44 -THS

lowast

lowast

lowastlowast

lowast

lowast

(e)

0

50

100

150

200SKOV-3

Con RVT 33 44 -THS

lowast lowast

(f)

0

50

100

150

200

CAT

activ

ity (

)

A2780

Con RVT 33 44 -THS

lowast

(g)

0

50

100

150

200OVCAR-3

Con RVT 33 44 -THS


lowastlowast

(h)

0

50

100

150

200SKOV-3

Con RVT 33 44 -THS

lowast

lowast

lowastlowast

(i)

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M



















0

50

100

150

2008-

OH

-dG

leve

l (

)A2780

lowast lowast

Con RVT 33 44 -THS

lowast lowast

(a)

0

50

100

150

200OVCAR-3

lowast

Con RVT 33 44 -THS

lowastlowastlowast

(b)

0

50

100

150

200SKOV-3

lowastlowast

Con RVT 33 44 -THS

lowastlowast

lowast

(c)

0

50

100

150

200

8-O

H-d

G le

vel (

)

RVT

A2780


lowastlowast

minus24 0 +24

Stan

dard

med

ium

(d)

0

50

100

150

200OVCAR-3

lowastlowastlowast

lowast

lowastlowast

RVT

Stan

dard

med

ium

minus24 0 +24

(e)

0

50

100

150

200SKOV-3


lowastlowast

lowast

RVT

Stan

dard

med

ium

minus24 0 +24

(f)


(g) (h) (i)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400

10 120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

0

1

2

3

4A2780

lowast lowast

Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

Time 0 +24

Stan

dard

med

ium

RVT

33

998400 44

998400

hOgg

1G

APD

H(m

RNA

OD

timesm

m2)

lowast lowast

0

1

2

3

4OVCAR-3

lowast lowast

lowast

Time 0Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

+24

Stan

dard

med

ium

RVT

33

998400 44

998400

lowastlowast

0

1

2

3

4SKOV-3

lowast lowastlowast

Time 0Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

Stan

dard

med

ium

RVT

33

998400 44

998400

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

+24

lowast lowast

Figure 4 Continued


GAPDH

hOgg1

hOgg1

10 1005010

(j)

10050

Time 0

RVT (120583M) 33998400 44998400-THS (120583M)

+24 hours











0

20

40

60

80

100

Apop

totic

cells

()

A2780

Con RVT

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100OVCAR-3

Con RVT

lowast

lowastlowastlowast

(b)

0

20

40

60

80

100SKOV-3

Con RVT

lowastlowast

lowast

lowast

lowast

(c)

(d)

0

50

100

150

200

250

Caspase-3

Caspase-9Caspase-8

Casp

ase a

ctiv

ity (

)

A2780

10050101005010C


lowast lowast

lowast

lowast

(e)

0

50

100

150

200OVCAR-3

Caspase-3

Caspase-9Caspase-8

10050101005010C


lowastlowast

lowast

lowast

(f)

0

50

100

150

200SKOV-3

Caspase-3

Caspase-9Caspase-8

10050101005010C

lowastlowast

lowastlowast

lowastlowastlowast

lowast

(g)

10120583M

100120583M50120583M

33998400 44998400-THS

10120583M

100120583M50120583M

33998400 44998400-THS

10120583M

100120583M50120583M

33998400 44998400-THS








0

25

50

75

100

125

150

Cel

l pro

lifer

atio

n (

)A2780

lowast

lowast lowastlowast

lowast

(a)

0

25

50

75

100

125

150

OVCAR-3

lowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

SKOV-3

lowastlowast

lowast

lowastlowastlowast

(c)

0

50

100

150

200

250A2780

lowastlowastlowast

(d)

0

100

200

300

400

500OVCAR-3

lowastlowast

lowast

lowast

lowast lowast

(e)

0

100

200

300

400SKOV-3

lowastlowast

lowastlowastlowast

lowast

(f)

(g)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THS


SA-120573

-Gal

activ

ity (

)





4 Discussion



0

50

100

150

200

Phos

phor

ylat

ed p

38to

tal p

38 (

) A2780

lowastlowast

lowast

(a)

0

25

50

75

100

125

150OVCAR-3

lowastlowast

(b)

0

25

50

75

100

125

150SKOV-3

lowast lowast

(c)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS




Low IC50



















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































0

50

100

150

2008-

OH

-dG

leve

l (

)A2780

lowast lowast

Con RVT 33 44 -THS

lowast lowast

(a)

0

50

100

150

200OVCAR-3

lowast

Con RVT 33 44 -THS

lowastlowastlowast

(b)

0

50

100

150

200SKOV-3

lowastlowast

Con RVT 33 44 -THS

lowastlowast

lowast

(c)

0

50

100

150

200

8-O

H-d

G le

vel (

)

RVT

A2780


lowastlowast

minus24 0 +24

Stan

dard

med

ium

(d)

0

50

100

150

200OVCAR-3

lowastlowastlowast

lowast

lowastlowast

RVT

Stan

dard

med

ium

minus24 0 +24

(e)

0

50

100

150

200SKOV-3


lowastlowast

lowast

RVT

Stan

dard

med

ium

minus24 0 +24

(f)


(g) (h) (i)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

33

998400 44

998400

10 120583M

100120583M50120583M

10120583M

100120583M50120583M

10120583M

100120583M50120583M

0

1

2

3

4A2780

lowast lowast

Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

Time 0 +24

Stan

dard

med

ium

RVT

33

998400 44

998400

hOgg

1G

APD

H(m

RNA

OD

timesm

m2)

lowast lowast

0

1

2

3

4OVCAR-3

lowast lowast

lowast

Time 0Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

+24

Stan

dard

med

ium

RVT

33

998400 44

998400

lowastlowast

0

1

2

3

4SKOV-3

lowast lowastlowast

Time 0Time (hours)

RVT 10120583M

RVT 100120583MRVT 50120583M

33998400 44998400-THS 10120583M

Stan

dard

med

ium

RVT

33

998400 44

998400

33998400 44998400-THS 100120583M33998400 44998400-THS 50120583M

+24

lowast lowast

Figure 4 Continued


GAPDH

hOgg1

hOgg1

10 1005010

(j)

10050

Time 0

RVT (120583M) 33998400 44998400-THS (120583M)

+24 hours











0

20

40

60

80

100

Apop

totic

cells

()

A2780

Con RVT

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100OVCAR-3

Con RVT

lowast

lowastlowastlowast

(b)

0

20

40

60

80

100SKOV-3

Con RVT

lowastlowast

lowast

lowast

lowast

(c)

(d)

0

50

100

150

200

250

Caspase-3

Caspase-9Caspase-8

Casp

ase a

ctiv

ity (

)

A2780

10050101005010C


lowast lowast

lowast

lowast

(e)

0

50

100

150

200OVCAR-3

Caspase-3

Caspase-9Caspase-8

10050101005010C


lowastlowast

lowast

lowast

(f)

0

50

100

150

200SKOV-3

Caspase-3

Caspase-9Caspase-8

10050101005010C

lowastlowast

lowastlowast

lowastlowastlowast

lowast

(g)

10120583M

100120583M50120583M

33998400 44998400-THS

10120583M

100120583M50120583M

33998400 44998400-THS

10120583M

100120583M50120583M

33998400 44998400-THS








0

25

50

75

100

125

150

Cel

l pro

lifer

atio

n (

)A2780

lowast

lowast lowastlowast

lowast

(a)

0

25

50

75

100

125

150

OVCAR-3

lowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

SKOV-3

lowastlowast

lowast

lowastlowastlowast

(c)

0

50

100

150

200

250A2780

lowastlowastlowast

(d)

0

100

200

300

400

500OVCAR-3

lowastlowast

lowast

lowast

lowast lowast

(e)

0

100

200

300

400SKOV-3

lowastlowast

lowastlowastlowast

lowast

(f)

(g)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THS


SA-120573

-Gal

activ

ity (

)





4 Discussion



0

50

100

150

200

Phos

phor

ylat

ed p

38to

tal p

38 (

) A2780

lowastlowast

lowast

(a)

0

25

50

75

100

125

150OVCAR-3

lowastlowast

(b)

0

25

50

75

100

125

150SKOV-3

lowast lowast

(c)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS




Low IC50



















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GAPDH

hOgg1

hOgg1

10 1005010

(j)

10050

Time 0

RVT (120583M) 33998400 44998400-THS (120583M)

+24 hours











0

20

40

60

80

100

Apop

totic

cells

()

A2780

Con RVT

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100OVCAR-3

Con RVT

lowast

lowastlowastlowast

(b)

0

20

40

60

80

100SKOV-3

Con RVT

lowastlowast

lowast

lowast

lowast

(c)

(d)

0

50

100

150

200

250

Caspase-3

Caspase-9Caspase-8

Casp

ase a

ctiv

ity (

)

A2780

10050101005010C


lowast lowast

lowast

lowast

(e)

0

50

100

150

200OVCAR-3

Caspase-3

Caspase-9Caspase-8

10050101005010C


lowastlowast

lowast

lowast

(f)

0

50

100

150

200SKOV-3

Caspase-3

Caspase-9Caspase-8

10050101005010C

lowastlowast

lowastlowast

lowastlowastlowast

lowast

(g)

10120583M

100120583M50120583M

33998400 44998400-THS

10120583M

100120583M50120583M

33998400 44998400-THS

10120583M

100120583M50120583M

33998400 44998400-THS








0

25

50

75

100

125

150

Cel

l pro

lifer

atio

n (

)A2780

lowast

lowast lowastlowast

lowast

(a)

0

25

50

75

100

125

150

OVCAR-3

lowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

SKOV-3

lowastlowast

lowast

lowastlowastlowast

(c)

0

50

100

150

200

250A2780

lowastlowastlowast

(d)

0

100

200

300

400

500OVCAR-3

lowastlowast

lowast

lowast

lowast lowast

(e)

0

100

200

300

400SKOV-3

lowastlowast

lowastlowastlowast

lowast

(f)

(g)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THS


SA-120573

-Gal

activ

ity (

)





4 Discussion



0

50

100

150

200

Phos

phor

ylat

ed p

38to

tal p

38 (

) A2780

lowastlowast

lowast

(a)

0

25

50

75

100

125

150OVCAR-3

lowastlowast

(b)

0

25

50

75

100

125

150SKOV-3

lowast lowast

(c)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS




Low IC50



















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

Apop

totic

cells

()

A2780

Con RVT

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100OVCAR-3

Con RVT

lowast

lowastlowastlowast

(b)

0

20

40

60

80

100SKOV-3

Con RVT

lowastlowast

lowast

lowast

lowast

(c)

(d)

0

50

100

150

200

250

Caspase-3

Caspase-9Caspase-8

Casp

ase a

ctiv

ity (

)

A2780

10050101005010C


lowast lowast

lowast

lowast

(e)

0

50

100

150

200OVCAR-3

Caspase-3

Caspase-9Caspase-8

10050101005010C


lowastlowast

lowast

lowast

(f)

0

50

100

150

200SKOV-3

Caspase-3

Caspase-9Caspase-8

10050101005010C

lowastlowast

lowastlowast

lowastlowastlowast

lowast

(g)

10120583M

100120583M50120583M

33998400 44998400-THS

10120583M

100120583M50120583M

33998400 44998400-THS

10120583M

100120583M50120583M

33998400 44998400-THS








0

25

50

75

100

125

150

Cel

l pro

lifer

atio

n (

)A2780

lowast

lowast lowastlowast

lowast

(a)

0

25

50

75

100

125

150

OVCAR-3

lowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

SKOV-3

lowastlowast

lowast

lowastlowastlowast

(c)

0

50

100

150

200

250A2780

lowastlowastlowast

(d)

0

100

200

300

400

500OVCAR-3

lowastlowast

lowast

lowast

lowast lowast

(e)

0

100

200

300

400SKOV-3

lowastlowast

lowastlowastlowast

lowast

(f)

(g)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THS


SA-120573

-Gal

activ

ity (

)





4 Discussion



0

50

100

150

200

Phos

phor

ylat

ed p

38to

tal p

38 (

) A2780

lowastlowast

lowast

(a)

0

25

50

75

100

125

150OVCAR-3

lowastlowast

(b)

0

25

50

75

100

125

150SKOV-3

lowast lowast

(c)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS




Low IC50



















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

25

50

75

100

125

150

Cel

l pro

lifer

atio

n (

)A2780

lowast

lowast lowastlowast

lowast

(a)

0

25

50

75

100

125

150

OVCAR-3

lowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

SKOV-3

lowastlowast

lowast

lowastlowastlowast

(c)

0

50

100

150

200

250A2780

lowastlowastlowast

(d)

0

100

200

300

400

500OVCAR-3

lowastlowast

lowast

lowast

lowast lowast

(e)

0

100

200

300

400SKOV-3

lowastlowast

lowastlowastlowast

lowast

(f)

(g)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THSCon RVT

10120583M

100120583M50120583M

33998400 44998400-THS


SA-120573

-Gal

activ

ity (

)





4 Discussion



0

50

100

150

200

Phos

phor

ylat

ed p

38to

tal p

38 (

) A2780

lowastlowast

lowast

(a)

0

25

50

75

100

125

150OVCAR-3

lowastlowast

(b)

0

25

50

75

100

125

150SKOV-3

lowast lowast

(c)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS




Low IC50



















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

Phos

phor

ylat

ed p

38to

tal p

38 (

) A2780

lowastlowast

lowast

(a)

0

25

50

75

100

125

150OVCAR-3

lowastlowast

(b)

0

25

50

75

100

125

150SKOV-3

lowast lowast

(c)

Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS Con RVT

10120583M

100120583M50120583M

33998400 44998400-THS




Low IC50



















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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