Research Article Synergic Effect of -Mangostin on the ...downloads.hindawi.com/journals/omcl/2016/7981397.pdfect against CDDP-induced nephrotoxicity, as well as antioxidant, antitumor,

Research ArticleSynergic Effect of 120572-Mangostin on the Cytotoxicity ofCisplatin in a Cervical Cancer Model

Jazmin M Peacuterez-Rojas1 Raquel Gonzaacutelez-Maciacuteas1 Jaime Gonzaacutelez-Cortes1

Rafael Jurado1 Joseacute Pedraza-Chaverri2 and Patricia Garciacutea-Loacutepez1

1Division de Investigacion Basica Instituto Nacional de Cancerologıa Av San Fernando 22 Apartado Postal 22026Tlalpan 14000 Mexico City Mexico2Facultad de Quımica Universidad Nacional Autonoma de Mexico 04510 Mexico City Mexico

Correspondence should be addressed to Jazmin M Perez-Rojas jazminmarlengmailcom

Received 24 June 2016 Revised 3 November 2016 Accepted 8 November 2016

Academic Editor Valentina Pallottini

Copyright copy 2016 Jazmin M Perez-Rojas et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Cervical cancer is the second leading cause of death among Mexican women The treatment with cis-diamminedichloroplatinum(II) (CDDP) has some serious side effects Alpha-mangostin (120572-M) has a protective effect against CDDP-induced nephrotoxicityas well as antioxidant antitumor and anti-inflammatory properties Hence we explored the in vitro and in vivo effect of 120572-Mon human cervical cancer cell proliferation when combined with CDDP In vitro The cytotoxic effect of 120572-M andor CDDPwas measured by the 3-(35-dimethylthiazol-2-yl)25-diphenyltetrazolium assay Meanwhile apoptosis reactive oxygen species(ROS) production and the cell cycle were determined with flow cytometry For 120572-M+CDDP treatment both a coincubation andpreincubation scheme were employed In vivo xenotransplantation was performed in female athymic BALBc (nunu) mice andthen tumor volume and body weight were measured weekly whereas 120572-M interfered with the antiproliferative activity of CDDPin the coincubation scheme with preincubation with 120572-M+CDDP showing significantly greater cytotoxicity than CDDP or 120572-M alone significantly inhibiting average tumor volume and preventing nephrotoxicity This effect was accompanied by increasedapoptosis and ROS production by HeLa cervical cancer cells as well as an arrest in the cell cycle These results suggest that 120572-Mmay be useful as a neoadjuvant agent in cervical cancer therapy

1 Introduction

Despite efforts to improve early detection of cervical cancerit is still one of the leading causes of death worldwide forwomen over 20 years of age In Mexico annually thereare about 68000 cases [1 2] Patients with this kind ofcancer have three options for treatment surgery radiother-apy chemotherapy or a combination In chemotherapy cis-diamminedichloroplatinum II (CDDP) is the most com-monly used drug [3] CDDP forms adducts between nitrogenbases induces apoptosis and generates reactive oxygen species(ROS) [4 5] Unfortunately CDDP has several secondaryeffects mainly in the kidney and brain and leads to chemore-sistance [6 7]Therefore it is necessary to continue searchingfor new anticancer compounds andor adjuncts to currenttreatments

Nowadays people consume many natural products toobtain beneficial health effects from their bioactive com-pounds One example is 120572-mangostin (120572-M) a prenylatedxanthone isolated from the mangosteen tree (Garcinia man-gostana Linn) Many in vitro and in vivo studies carriedout on 120572-M have reported antioxidant and antitumorigeniceffects among other properties [8 9] In the last few yearsstudies on the anticancer properties of 120572-M and othercompounds derived from the mangosteen tree have shownexcellent results mostly demonstrating their efficacy and lowtoxicity

Since the sale of juices and extracts of these compoundsis not restricted they are freely consumed Sometimespatients under chemotherapy use such products unaware ofthe effect that may result from combining them with theirtreatment There are few studies on the possible impact of

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 7981397 13 pageshttpdxdoiorg10115520167981397

2 Oxidative Medicine and Cellular Longevity

the natural compounds from the mangosteen tree on theantiproliferative and secondary effects produced by CDDPHence we decide to conduct such a study with 120572-M becauseit is known to improve the antiproliferative effect of CDDPwhile minimizing its secondary effects [9ndash11] At the sametime it minimally alters normal cells [12ndash14]

Previously we demonstrated that 120572-M has a renopro-tective effect on CDDP-induced nephrotoxicity This effectwas mediated by preventing an increase in ROS tumornecrosis factor-120572 and transforming growth factor 120573 withoutshowing changes in the pharmacokinetics of CDDP [15]Since 120572-M has antiproliferative and renoprotective effectsthe aim of the present study was to evaluate the possibilitythat the combination of this compound with CDDP couldimprove cytotoxicity and diminish secondary effects Thuswe determined the cytotoxic effect of CDDP+120572-M in vitro aswell as in an in vivo cervical cancer model

2 Material and Methods

21Materials 120572-Mwas purified as previously described frommangosteen pericarp [16] purchased from DNP Interna-tional Inc (Whittier CA USA) CDDP 3-(35-dimethylthi-azol-2-yl)25-diphenyltetrazolium (MTT) and 5-(and-6)-carboxy-2101584071015840-dichlorodihydrofluorescein diacetate (car-boxy-H2DCFDA) were obtained from Sigma-Aldrich Co(St Louis MO USA) Dimethyl sulfoxide (DMSO) was ob-tained from MP Biomedicals (Solon OH USA) All rea-gents for flow cytometry were obtained from Millipore Inc(Darmstadt DE USA) All other reagents usedwere obtainedfrom commercial sources

22 Cell Culture Thehuman cervix adenocarcinoma cell line(HeLa)was obtained fromAmericanTypeCulture Collection(Manassas VA USA) Cells were routinely maintained inDulbeccorsquos modified Eaglersquos medium (DMEM) supplementedwith 10 fetal bovine serum (Gibco BRL) and incubated at37∘C under an atmosphere of 5 CO2 at high humidity

23 Cell Viability Assay Mitochondrial function was esti-mated by the MTT assay which is based on the reduction of atetrazolium salt by the mitochondrial dehydrogenase enzymein viable cells After treatment the medium was removed5mgmL MTT was diluted in DMEM without phenol red(1 9) and 100120583L of reagent was added and then the solutionwas incubated for 1 h Formazan dye was dissolved with 2-propanol acid and optical density was measured using anELISA reader at 120582 570nm The results are expressed as thepercentage of MTT reduction in relation to control values

24 Effect of 120572-M and CDDP on Cell Viability The effect of120572-M and CDDP on cell viability was determined using theMTT assay After seeding 1times104 cellswell in culture mediumin 96-well plates 120572-M (0 to 80 120583M) or CDDP (0 to 120 120583M)was added cells were incubated for 24 h and finally cellviability was quantified The percentage of growth inhibitionwas calculated and the concentration was determined atwhich each drug achieved 50 growth inhibition (IC50) Atheoretical isobologram was then constructed to determine

the concentration that would be used for the drug com-binations subsequently evaluated in two different schemescoincubation and preincubation Whereas in the formerscheme both agents were coincubated for 24 h in the latterthe cells were exposed to 120572-M for 24 h and then incubatedwith CDDP for an additional 24 h The results are expressedas the percentage of MTT reduction The experiment wasconducted in triplicate in independent experiments

25 Isobologram The isobologram method was used todetermine the inhibition of cell viability in function of theinteraction between the two drugs [17 18]The IC50 of CDDPalone was plotted on the abscissa and of 120572-M on the ordinateThe combination of these two compounds having additiveeffects should fall on a straight line connecting these twopoints While points above the line represent antagonismthose below the line show synergism

26 Drug Interaction Analysis The combination index (CI)calculated for data analysis of combined drug treatmentwas determined by median-effect principle derived by Chou[19 20] The equation correlates the drug dose and cytotoxiceffect in the following way CI = (11986311198631199091) + (11986321198631199092) +120572(119863111986321198631199091 lowast1198631199092) where1198631 and1198632 represent the concen-trations used in the combined treatment while 1198631199091 and 1198631199092are single treatment concentrations giving the same responseas 1198631 and 1198632 respectively The factor 120572 indicates the typeof interaction 120572 = 0 for similar mechanisms of actionand 120572 = 1 for independent modes of action (120572 = 1 washerein employed) CI = 1 indicates an additive effect CI lt1 synergism and CI gt 1 antagonism

27 Determination of Reactive Oxygen Species (ROS) Pro-duction by Flow Cytometry Cells (2 times 105) were seededin 6-well plates and then treated by using the coincuba-tion or preincubation scheme The cells were later washedwith phosphate-buffered saline solution (PBS) and harvestedwith PBS-EDTA followed by centrifuging at 2000 rpm for8 minutes and resuspension in PBS For ROS detectionthey were treated with 10120583M of the fluorescent marker 5-(and-6)-carboxy-2101584071015840-dichlorodihydrofluorescein diacetate(carboxy-H2DCFDA Sigma-Aldrich) [21] for 30 minutesin the dark Flow cytometry was performed over 10000acquired events with InCyte software (Merck MilliporeDarmstadt DE) with hydrogen peroxide (1mM per 24 h)used as the positive control The experiment was carried outin triplicate

28 Determination of Apoptosis by Flow Cytometry Cells(2 times 105) were seeded in 6-well plates and then treatedby using the coincubation or preincubation scheme Thecells were later washed with PBS and harvested with PBS-EDTA followed by centrifugation at 2000 rpm for 8minutesCells were resuspended in PBS and then treated with GuavaNexin Reagent to measure apoptosis at room temperaturefor 20 minutes in the dark Flow cytometry was performedover 10000 acquired events with Guava Nexin software(Merck Millipore Darmstadt DE) Early and late apoptosiswas determined and the results are expressed as the total

Oxidative Medicine and Cellular Longevity 3

percentage of apoptosis Experiments were carried out intriplicate

29 Determination of the Cell the Cycle by Flow CytometryCells (2times 105) were seeded in 6-well plates and then treated byusing the coincubation or preincubation scheme Afterwardsthe cells were washed with PBS and harvested with PBS-EDTA followed by centrifugation at 2000 rpm for 8minutesThey were then resuspended in PBS and 70 ethanol andincubated at 4∘C overnight Thereafter to measure the cellcycle cells were washed with PBS and then treated withGuavaCell Cycle Reagent at room temperature for 30minutesin the dark Flow cytometry was performed over 10000acquired events with cellCycle software (Merck MilliporeDarmstadt DE) and cell cycle phases were analyzed Exper-iments were carried out in triplicate

210 In Vivo Experiments Female BALBc mice (nunu 20ndash25 g body weight) were provided by UPEALvivarium ofMetropolitan University (Mexico City Mexico) The pro-cedures for animal care and use were approved by theEthics Committee of the Instituto Nacional de Cancerologıa(INCan Mexico City Mexico) The mice had free accessto sterile water and food and were kept in a pathogen-free environment (Allentown Inc USA) at 25∘C and 70humidity All mice were handled in accordance with theMexican Guide (NOM-062-ZOO-1999) and the Committeefor Updating the Guide for the Care and Use of LaboratoryAnimals [22] Biological hazardous residues were discardedaccording to the corresponding guide (NOM-087-ECOL-SSA1-2001)

211 Experimental Design All mice were subcutaneouslyinoculated in the back with 5 times 106 HeLa (cervical cancer)cells Once the tumor reached about 150mm3 mice weredivided into the following four groups (1) control orallyadministrated 05 carboxymethyl-cellulose (2) the 120572-Mtreatment was treated orally with 125mgKg (suspendedin 05 carboxymethyl-cellulose) (3) the CDDP treatmentintraperitoneally injected with CDDP (3mgKg) and (4) theCDDP+120572-M treatment administered at the doses and bythe routes aforementioned for these compounds 120572-M wasadministrated for four days before injecting CDDP once aweek for four weeks 120572-M and CDDP dose used in the presentstudy was chosen according to previous experiments per-formed in the laboratory [15 23]The tumors were measuredin two dimensions with a caliper and tumor volume wascalculated by the following equation 119881 = (1205876)((width)2 lowastlength) [24] Cell doubling time (CDT) was calculated by thefollowing formula CDT = (days of treatment)((Log (finaltumor) ndash Log (initial tumor))Log 2) [25 26] Tumor andbody weight were measured once a week for 10 weeks Atthe end of the experiment the mice were placed in metaboliccages for 24 h after which time animals were anaesthetizedwith a mix of isofluraneoxygen 3 Blood and urine samplewere collected and frozen at minus80∘C to await processing

212 Renal Toxicity Blood urea nitrogen (BUN) levels weremeasured with an autoanalyzer (Beckman Coulter labora-

tory analyzer AU680 Chemistry System) Urine protein wasanalyzed by using the bicinchoninic acid assay [27] withbovine serum albumin (BSA) as the standard Kidney injurymolecule 1 (KIM-1) was measure with an immunoassay kit(Cloud-Clone Corp TX USA) following the manufacturerrsquosinstructions

213 Statistics Values are expressed as the mean plusmn SD ofat least three independent experiments The tumor volumewas analyzed by ANOVA for repeated measures followedby the Student Newman-Keuls comparison test The otherparameters were evaluated using one-way ANOVA and theStudent Newman-Keuls correction for multiple comparisonswith GraphPad Prism 4 software (San Diego CA) Differ-ences were considered significant at 119901 le 005

3 Results

31 Cytotoxicity of 120572-M andCDDPonHumanCervical CancerCells The IC50 for CDDP was 297 plusmn 13 120583M and for 120572-M191 plusmn 19 120583M With these data an isobologram was built toobtain the concentrations for the combined drug treatmentthat should give an additive effect as well as those thattheoretically give a synergist effect (data not shown)

The cytotoxic effect of CDDP in HeLa cells is depictedin Figure 1 for coincubation (Figure 1(a)) and preincubation(Figure 1(b)) CDDP and 120572-M showed a cytotoxic effect ina concentration-dependent manner For the coincubationscheme the combined treatment with 120572-M+CDDP providedgreater protection of the cell against CDDP cytotoxicity atlow concentrations of 120572-M (5 to 15120583M) compared to thehigh concentration This effect of the combined treatment at5 to 15120583M of 120572-M was statically significant in relation to theCDDP treatment alone In the preincubation scheme on theother hand the combined treatment decreased cell viabilityin a concentration-dependent manner for both compoundsMoreover the concentration of 10 and 15120583M of 120572-M alsoshowed statistical significance with respect to the CDDPtreatment alone (Figure 1(b))

32 Drug Interaction of 120572-M and CDDP Once the IC50was obtained for 120572-M and CDDP separately the effect ofthe combined treatment on the percentage of cell viabilitywas determined In the coincubation scheme neither ofthe combinations produced a synergistic effect With thepreincubation scheme contrarily 120572-M increased CDDP cyto-toxicity in HeLa cells (Table 1) Based on these results wechose the concentration of 10 and 15120583M of 120572-M and 2 120583Mof CDDP for subsequent experiments For both the preincu-bation and coincubation schemes the higher concentrationsof 120572-M (25120583M or greater) combined with any concentrationof CDDP led to a cell viability that was not different fromtreatment with 120572-M alone (Figure 1)

33 Effect of 120572-M and CDDP on ROS Production In the coin-cubation scheme no concentration of 120572-M or CDDP alonenor any combined treatment could significantly increase ROSproduction (Figure 2) With the preincubation scheme the


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Figure 1 Cell viability Using the combined treatment with 120572-M+CDDP the cytotoxic effect on HeLa human cervical cancer cells wasdetermined with (a) the coincubation scheme and (b) the preincubation schemeThe values are expressed as a percentage of viability (100)found in the absence of the drug Each point shows the mean plusmn SD of three independent experiments A versus CDDP group 119901 le 005

Table 1 Summary of the effect on HeLa cell of CDDP 120572-M and their combinations in the preincubation scheme The table presents thecombination index (CI) calculated based on the equation given in the text lowastMean values of three separate experiments performed in triplicate

Drugs in combination Drugs alone Control growth ()lowast Combination index InteractionCDDP 120583M (D1) 120572-M 120583M (D2) CDDP 120583M (1198631199091) 120572-M 120583M (1198631199092)2 5 66 84 898 112 10 207 138 730 08 Synergistic5 10 254 148 680 10 Additive2 15 379 165 561 10 Additive

combination of 2120583M of CDDP plus 15 120583M of 120572-M signif-icantly elevated ROS generation compared to the controlgroup and the individual treatments (Figure 3)

34 Effect of 120572-M and CDDP on Apoptosis In the apoptosisanalysis we did not find a statistically significant differencebetween any of the groups in the coincubation scheme(Figure 4) In the preincubation scheme however both com-bined treatments significantly increased the percentage ofapoptosis in relation to the control group (Figure 5) In factthe combination of CDDP and 15120583Mof 120572-M is different fromthe treatment with 120572-M alone at this same concentrationThese data correlate with the decrease in cell viability foundwhen using this combined treatment in the preincubationscheme

35 Effect of 120572-M and CDDP on the Cell Cycle The dis-tribution of the cell cycle was analyzed when cells weretreated with 120572-M CDDP and 120572-M+CDDP (considering thecoincubation and preincubation schemes) (Table 2) CDDPalone increased the S and G2M phase although it only wassignificant in the preincubation scheme Contrarily 120572-M at

both concentrations gave similar results to those found in thecontrol group On the other hand the combined treatmentsincreased the arrest in G2M phase like the CDDP group butin some cases did not reach statistical significance

36 In Vivo Effect of the 120572-M+CDDP Treatment Uponfinding that 120572-M had a synergistic effect with CDDP incell cultures and that the pretreatment scheme was moreeffective we decided to conduct experiments on mice usingonly this scheme The combined treatment proved to bemore effective for controlling the tumor growth rate than thevehicle (control) and the individual treatments (Figure 6(a))Additionally for mice treated with the combined treatmentversus animals in the control group the mean cell doublingtime in tumors was 9 days versus 4 days (Table 3) a dif-ference that was statistically significant We then measuredsystemic and renal toxicity in all groups finding no signif-icant difference in the body weight of mice between any ofthe studied groups (Figure 6(b)) Moreover there were nosignificant changes in the BUN levels between any of thestudied groups (data not show) Treatment with CDDP alonesignificantly increased urinary volume urinary protein and


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KIM-1 compared to the control Conversely 120572-M+CDDPtreatment led to no increase in those parameters (Figures 7(b)and 7(c))

4 Discussion

Cervical cancer is a worldwide public health problem CDDPis the gold standard of chemotherapy for this type of canceralthough it has several side effects Therefore new drugsandor modalities of treatment should be explored [28]

Over recent years in Mexico and around the world therehas been an increase in cancer research and a greater focus

on new therapeutic strategies and detection methods [29] Ithas been reported that a therapeutic and preventive effect canbe achieved for several types of cancer with phytochemicalderivatives of food or food sources like capsaicin (peppers)[30] curcumin (curcuma) [31] resveratrol (grapes) [32]lycopene (tomatoes) [33] cinnamon essential oil [34] andothers [35]

Regarding 120572-M it is known to have antioxidant anti-tumorigenic anti-inflammatory and antibacterial properties[8] This compound has been under study in the las fewyears because of these properties as well as its antiproliferativeeffect It has been demonstrated that 120572-M decreases cellular


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Figure 3 ROS generation (a) Flow cytometry of ROS production with representative histograms The empty histogram denotes the controlgroup (b)The effect of CDDP 120572-M and the combined treatments on ROS generation with the preincubation scheme Each bar represents themeanplusmn SDof four independent experiments A versus control group B versusCDDPgroup C versus120572-M 15120583MgroupD versusCDDP+120572-M210 group 119901 le 005

proliferation in vivo and in vitro with leukemia [36ndash38]colon cancer [12 39] prostate cancer [13] and breast cancer[40] Moreover our group previously reported that 120572-M hasa renoprotective effect against damage induced by CDDPnephrotoxicity [15] To our knowledge the possible adjuvanteffect of 120572-M when combined with CDDP has not beenpreviously described

The perfect combination of drugs would be one gen-erating synergism against cancer cells without increasingsystemic toxicity A synergistic effect refers to a combination

of drugs whose effect is numerically better than that obtainedby either of its components used individually [18] The USFood and Drug Administration (FDA) has already approvedthe combination of CDDP with adjuvant drugs to improvethe efficacy of treatment and the health of patients [41]

The mechanism of action of CDDP in the cell is theformation of platinum-DNA adducts and the inhibitionof cell replication and transcription provoking cell cyclearrest and then cell death [42] Additionally CDDP causesapoptotic cell death in the proximal tubular cell which has


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been attributed to the generation of ROS [14] In the presentstudy we demonstrated that CDDP decreased cell viability ina dose-dependent manner (Figure 1) and induced cell cyclearrest in the G2M phase (Table 2)

The mechanism of cell death stimulated by 120572-M isnot completely clear Among the mechanisms reported is

the inhibitory effect on human topoisomerases I and IIproteins that are necessary for chromosome segregation inthe daughter cell Thus 120572-M suppresses cell proliferationleading cells to apoptosis [43] Another mechanism is theinhibition of CDK4 kinase which restricts progression ofthe cell cycle [13] 120572-M has also been associated with cell


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cycle arrest in the G2M phase by regulating expression ofcdc2 cyclin and p27 [44] Recently Aisha and coworkers [45]found that 120572-M induces apoptosis by several mechanismssuch as through the MycMax and MAPKERK signalingpathways and the downregulation of the NFkB pathway In

this study we designed two experimental schemes to studyseveral mechanisms for both drugs

We established that the IC50 of 120572-M in HeLa culturecells was 197 plusmn 10 120583M similar to the value determined byMizushina and coworkers [43] This value is lower than the


Table 2 Cell cycle analysis after treatment with 120572-M CDDP and the combined treatments using both the coincubation and preincubationschemes Values are the mean plusmn SD of three independent experiments for each point

Groups G1G0 S G2M G2G1 ratioCoincubation

Control 591 plusmn 63 133 plusmn 43 276 plusmn 48 048 plusmn 012

CDDP 2 120583M 427 plusmn 83a 233 plusmn 74a 340 plusmn 58 083 plusmn 026

120572-M 10120583M 624 plusmn 71 123 plusmn 28 253 plusmn 48 042 plusmn 013


CDDP+120572-M (210120583M) 428 plusmn 21a 185 plusmn 42 387 plusmn 47a 091 plusmn 014

CDDP+120572-M (215120583M) 602 plusmn 87 138 plusmn 46 260 plusmn 59 045 plusmn 015

PreincubationControl 515 plusmn 17 119 plusmn 15 366 plusmn 22 071 plusmn 006

CDDP 2 120583M 298 plusmn 61a 206 plusmn 49a 538 plusmn 60a 186 plusmn 041



CDDP+120572-M (210120583M) 272 plusmn 58a 225 plusmn 65a 453 plusmn 72 173 plusmn 043

CDDP+120572-M (215120583M) 401 plusmn 112a 232 plusmn 51a 388 plusmn 87 117 plusmn 063a119901 le 005 versus control group

Table 3 Growth response of cervical cancer tumors treatedwith thecombination of CDDP+120572-M Values are the mean plusmn SD of 6ndash8 miceper group

Tumor volume (mm3) Cell doubling time(days)Initial Final

Control 168 plusmn 59 1132 plusmn 445 39 plusmn 12

CDDP 144 plusmn 41 712 plusmn 303a 47 plusmn 12b

120572-M 143 plusmn 36 676 plusmn 325a 49 plusmn 23c

CDDP+120572-M 192 plusmn 93 371 plusmn 233a 85 plusmn 43a

aversus control group bversus control CDDP cversus 120572-M group 119901 le 005

IC50 for CDDP (297plusmn13 120583M)However when120572-Mwas coin-cubated with CDDP the percentage of cell viability did notdecrease more than 60 percent (Figure 1(a)) indicating thatlow concentrations of 120572-M protect the cells against CDDPdamage In this sense Aisha and coworkers [45] reportedthat at low concentrations 120572-M significantly reduced CDDPcytotoxicity on colorectal carcinoma cells However whenthe cells were exposed first to 120572-M and then CDDP theresponse was contrary At almost all concentrations testedthe percentage of cell viabilitywas lower for the preincubationthan coincubation scheme (Figure 1(b)) After exposure tothe drugs the combination index was calculated (Table 1)

In the coincubation scheme 120572-M interferes with thecytotoxicity activity of CDDP which was probably the reasonthat changes were not observed in ROS generation In thepreincubation scheme most combinations showed an addi-tive effect These results suggest that at low concentrations120572-M only exerts cytotoxic effects if it is administered beforecancer cells are exposed to CDDP In both schemes anyconcentration of 120572-M up to EC50 kills cancer cells (Figure 1)This indicates the dominance of the cytotoxic effect inducedby 120572-M over CDDP

Upon finding that 120572-M enhanced the cytotoxicity ofCDDP we explore the possible mechanisms involved AsCDDP generates ROS and 120572-M is an antioxidant compoundROS production was measured There was a tendency toincreased ROS production (not reaching a statistical signifi-cance in relation to the control group) at a low concentrationof CDDP or 120572-M alone as well as with the combinationtreatment in the coincubation scheme (Figure 2) In thepreincubation scheme only the combination of 2 120583MCDDPplus 15120583M 120572-M resulted in a significant increase of ROSgeneration with respect to the control group (Figure 3)

The fact that 120572-M herein stimulated CDDP-inducedROS production seems contrary to previous reports onthis xanthone which has been described as a scavengerof several ROS in a concentration-dependent manner [46]Nevertheless the 15120583M concentration of 120572-M alone didnot elevate ROS levels According to Somasundaram andcoworkers [47] the combination of nitric oxide donors andROS generationmay have a bifunctional response promptinga pro- or antitumorigenic effect The authors posed that thisresponse depends on four elements (1) the concentrationof the NO donor and ROS inducers (2) the treatmentregimen (3) the duration of treatment and (4) the geneticsof the cancer cells Additionally Halliwell [48] indicated thatantioxidant agents might act as prooxidant compounds aspart of a mixture (eg the mixture of 120573-carotene ascorbateand 120572-tocopherol) Hence 120572-Mmay act as a prooxidant afterpreviously functioning as an antineoplastic agent and thencombined with CDDP at a low concentration

The combination of 120572-M and CDDP decreased theviability of cervical cancer cells (Figure 1) which correspondsto an additive effect (Table 1) Apart from a rise in ROSlevels (Figure 3) the combination of 120572-M and CDDP in thepreincubation scheme caused a higher percentage of apopto-sis The present results are similar to those reported in theliterature [13 42] (Figure 5) Although in the coincubation


A B C

Control

2 4 6 8 100Time (weeks)

A

CDDP (3mgKg)-M (125mgKg)

Tum

or v

olum

e (m

m3)

times102

0

5

10

15

20

CDDP+-M

(a)

ControlCDDP

-M

0

10

20

30

40

50

Body

wei

ght (

g)

2 4 6 8 100Time (weeks)

CDDP+-M

(b)

Figure 6 (a) Time course of tumor volume growth in mice after a subcutaneous implant of HeLa cells that were treated with the vehicle(control) CDDP 120572-M and CDDP+120572-M (b) Body weight measured once per week of mice treated with the vehicle CDDP 120572-M andCDDP+120572-M Each bar represents the mean plusmn SD of six to eight animals A versus control group B versus CDDP group C versus 120572-M group119901 le 005

A

Control CDDP -M0

1

2

3

4

5

CDDP+-M

Urin

ary

volu

me (

mL

24

h)

(a)Control CDDP -M

0

20

40

60

80

CDDP+-M

A B C

Urin

ary

prot

ein

(mg24

h)

(b)

A B C

Control CDDP -M0

500

1000

1500

2000

CDDP+-M

KIM

-1(p

gm

L)

(c)

Figure 7 Markers of nephrotoxicity for all groups (a) Urinary volume (b) Urinary protein (c) Kidney injury molecule 1 (KIM-1) CDDPcisplatin 120572-M alpha mangostin Each bar represents the mean plusmn SD of six to eight animals A versus control group B versus 120572-M group Cversus CDDP+120572-M group 119901 le 005


schemewe did not find a significant difference between any ofstudy groups there is evidence in the literature indicating that120572-M induces apoptosis in YD-15 tongue mucoepidermoidcarcinoma cells starting at a concentration of 10 120583M [49]

In the preincubation scheme the combined treatment of120572-M+CDDP herein prompted cell cycle arrest in the G2Mphase (Table 2) as did CDDP administered alone Similarresults were found in an oral squamous cell line after 24 h ofexposure without any changes detected at 48 h [41] On theother hand for 120572-M alone we observed no changes in the cellcycle However it has been described that 120572-M significantlylengthens the duration of the G2M phase in a colon cancercell line [42] whereas in prostate cancer and pancreas cancer120572-M induces arrest in G1 [13 50] So the modification of thecell cycle by 120572-M depends on the cell line concentration andexposure time

With the combination of the two compounds 120572-Mseems to decrease the cell cycle arrest of CDDP in theG2M phase with the preincubation scheme which mayindicate interference by 120572M in the cytotoxic effect of CDDPNevertheless the present data on cell viability and apoptosisshow that the 120572M+CDDP combination kills the cancer cellsHence this combination probably activates other pathwaysnot necessarily involving alterations in the cell cycle Forexample it has been reported that 120572-M has effect on Ca2+-ATPase activity on endoplasmic reticulum and also causes aloss in the mitochondrial membrane potential and the releaseof cytochrome c leading to apoptosis in PC12 cells [51]Further experiments are needed to elucidate how a treatmentbased on 120572M and CDDP combination induces cell death

After obtaining the current in vitro results we pro-posed an experimental model of xenograft (Figure 6(a)) Wedemonstrated that the administration of 120572-M before CDDPdelayed tumor growth evidenced by a decrease in the finaltumor volumeThis antitumorigenic effect correlated with anincrease in the cell doubling time in that group (Table 3) anoutcome not accompanied by systemic toxicity (Figure 6(b))

Systemic toxicity and renal toxicity do not necessarilyparallel events The kidney has the capacity to compensate fora loss of function when some of its nephrons are damagedFor this reason in the present study we use some of earlymarkers of kidney damage such as BUN urinary volumeurinary protein andKIM-1The current results confirm thosefound previously in which 120572-M prove to protect the kidneyfrom CDDP-induced damage (Figure 7) without modifyingthe pharmacokinetic of this drug [15] It has previously beenreported that 120572-M is effective against several types of cancerincluding pancreas [50] prostate [13] and breast tumors [52]Weherein demonstrated for the first time that this compoundis effective in the treatment of cervical cancer Furthermoreevidence is presently provided in relation to the beneficialeffect resulting from the combination of a natural product (120572-M) and chemotherapy (CDDP) if the scheme of treatment isaccurate

In summary the treatment combining 120572-M and CDDPled to distinct outcomeswith a coincubation or preincubationscheme The combined and simultaneous administrationof 120572-M and CDDP caused a strong interaction between

the two drugs and protection of the cancer cells Con-trarily the administration of 120572-M before CDDP improvedthe therapeutic response exhibited by CDDP alone Thispreincubation scheme limited tumor volume growth andaugmented the cell doubling time without giving rise tosecondary effects (systemic damage andor nephrotoxicity)There was an increase in cell death ROS production andapoptosis as well as the arrest of the cell cycle Hence 120572-M pretreatment increased CDDP toxicity without producingsecondary effects The current data suggest that 120572-M can beused as an adjuvant agent in those cancers whose treatment isbased on CDDP However it is necessary to carry out clinicaltrials with patients to confirm the current findings

Additional Points

Highlights Preincubation with 120572-M increased CDDP cyto-toxicity in HeLa cultured cells The combination of 120572-M+CDDP stemmed tumor growth 120572-M avoids kidney damagewithout hindering with CDDPrsquos antitumorigenic effect

Competing Interests

The authors declare that there are no competing interests

Acknowledgments

This work was supported by the Mexican Council of Sci-ence and Technology (CONACYT) (research Grant 178946)Raquel Gonzalez-Macıas was supported by a scholarshipgrant (PROBEI) from the Health System of Mexico

References

[1] J M Walboomers M V Jacobs M M Manos et al ldquoHumanpapillomavirus is a necessary cause of invasive cervical cancerworldwiderdquo The Journal of Pathology vol 189 no 1 pp 12ndash191999

[2] Instituto Nacional de Estadıstica y Geografıa Dıa mundial con-tra el cancer [On line] httpwwwinegiorgmxinegidefaultaspxc=274

[3] R Fontanelli G Spatti F Raspagliesi F Zunino and FDi Re ldquoApreoperative single course of high-dose cisplatin and bleomycinwith glutathione protection in bulky stage IBII carcinoma ofthe cervixrdquo Annals of Oncology vol 3 no 2 pp 117ndash121 1992

[4] J C Garcıa-Ramos R Galindo-Murillo F Cortes-Guzmanand L Ruiz-Azuara ldquoMetal-based drug-DNA interactionsrdquoJournal of the Mexican Chemical Society vol 57 no 3 pp 245ndash259 2013

[5] S Jaiman A K Sharma K Singh and D Khanna ldquoSignallingmechanisms involved in renal pathological changes duringcisplatin-induced nephropathyrdquo European Journal of ClinicalPharmacology vol 69 no 11 pp 1863ndash1874 2013

[6] V CepedaMA Fuertes J Castilla C Alonso CQuevedo andJ M Perez ldquoBiochemical mechanisms of cisplatin cytotoxicityrdquoAnti-Cancer Agents in Medicinal Chemistry vol 7 no 1 pp 3ndash18 2007

[7] M A Fuertes C Alonso and J M Perez ldquoBiochemicalmodulation of cisplatin mechanisms of action enhancement


of antitumor activity and circumvention of drug resistancerdquoChemical Reviews vol 103 no 3 pp 645ndash662 2003

[8] J Pedraza-Chaverri N Cardenas-Rodrıguez M Orozco-Ibarra and J M Perez-Rojas ldquoMedicinal properties of man-gosteen (Garciniamangostana)rdquo Food andChemical Toxicologyvol 46 no 10 pp 3227ndash3239 2008

[9] S Jindarat ldquoXanthones from mangosteen (Garcinia man-gostana) multi-targeting pharmacological propertiesrdquo Journalof the Medical Association of Thailand vol 97 no S2 pp S196ndashS201 2014

[10] K Uehara and M Nagino ldquoNeoadjuvant treatment for locallyadvanced rectal cancer a systematic reviewrdquo Surgery Today vol46 no 2 pp 161ndash168 2016

[11] N Turner L Biganzoli and A Di Leo ldquoContinued value ofadjuvant anthracyclines as treatment for early breast cancerrdquoThe Lancet Oncology vol 16 no 7 pp e362ndashe369 2015

[12] A F A Aisha K M Abu-Salah Z Ismail and A MajidldquoIn vitro and in vivo anti-colon cancer effects of Garciniamangostana xanthones extractrdquo BMC Complementary andAlternative Medicine vol 12 article 104 2012

[13] J J Johnson S M Petiwala D N Syed et al ldquo120572-Mangostina xanthone from mangosteen fruit promotes cell cycle arrestin prostate cancer and decreases xenograft tumor growthrdquoCarcinogenesis vol 33 no 2 pp 413ndash419 2012

[14] Y Sanchez-Perez R Morales-Barcenas C M Garcıa-Cuellaret al ldquoThe 120572-mangostin prevention on cisplatin-induced apop-totic death in LLC-PK1 cells is associated to an inhibition ofROSproduction andp53 inductionrdquoChemico-Biological Interactionsvol 188 no 1 pp 144ndash150 2010

[15] J M Perez-Rojas C Cruz P Garcıa-Lopez et al ldquoRenopro-tection by 120572-mangostin is related to the attenuation in renaloxidativenitrosative stress induced by cisplatin nephrotoxicityrdquo Free Radical Research vol 43 no 11 pp 1122ndash1132 2009

[16] B Marquez-Valadez P D Maldonado S Galvan-Arzate etal ldquoAlpha-mangostin induces changes in glutathione levelsassociated with glutathione peroxidase activity in rat brainsynaptosomesrdquo Nutritional Neuroscience vol 15 no 5 pp 13ndash19 2012

[17] R J Tallarida F Porreca and A Cowan ldquoStatistical analysisof drug-drug and site-site interactions with isobologramsrdquo LifeSciences vol 45 no 11 pp 947ndash961 1989

[18] R J Tallarida ldquoCombination analysisrdquo Advances in Experimen-tal Medicine and Biology vol 678 pp 133ndash137 2010

[19] T-C Chou ldquoDrug combinations from laboratory to practicerdquoJournal of Laboratory and Clinical Medicine vol 132 no 1 pp6ndash8 1998

[20] T-C Chou ldquoDrug combination studies and their synergy quan-tification using theChou-TalalaymethodrdquoCancer Research vol70 no 2 pp 440ndash446 2010

[21] A Gomes E Fernandes and J L F C Lima ldquoFluorescenceprobes used for detection of reactive oxygen speciesrdquo Journalof Biochemical and Biophysical Methods vol 65 no 2-3 pp 45ndash80 2005

[22] Committee for the Update of the Guide for the Care and Use ofLaboratory Animals Guide for the Care and Use of LaboratoryAnimals The National Academies Press 2010

[23] M Segovia-Mendoza R Jurado R Mir L A Medina HPrado-Garcia and P Garcia-Lopez ldquoAntihormonal agents as astrategy to improve the effect of chemo-radiation in cervicalcancer In vitro and In vivo Studyrdquo BMC Cancer vol 15 noarticle 21 2015

[24] L-A Medina B-I Herrera-Penilla M-A Castro-Morales etal ldquoUse of an orthovoltage X-ray treatment unit as a radiationresearch system in a small-animal cancer modelrdquo Journal ofExperimental amp Clinical Cancer Research vol 27 no 1 article57 2008

[25] J K Smolev D S Coffey and W W Scott ldquoExperimentalmodels for the study of prostatic adenocarcinomardquo Journal ofUrology vol 118 no 1 1977

[26] T W Redding and A V Schally ldquoInhibition of prostate tumorgrowth in two rat models by chronic administration of D-Trp6 analogue of luteinizing hormone-releasing hormonerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 78 no 10 pp 6509ndash6512 1981

[27] P Yalamati A V Bhongir M Karra and S R Beedu ldquoCom-parative analysis of urinary total proteins by bicinchoninic acidand pyrogallol red molybdate methodsrdquo Journal of Clinical andDiagnostic Research vol 9 no 8 pp BC01ndashBC04 2015

[28] L Cetina L Rivera J Hinojosa et al ldquoRoutine management oflocally advanced cervical cancer with concurrent radiation andcisplatin Five-year resultsrdquo BMCWomenrsquos Health vol 6 article3 2006

[29] K Vassbakk-Brovold S Berntsen L Fegran et al ldquoIndividu-alized comprehensive lifestyle intervention in patients under-going chemotherapy with curative or palliative intent whoparticipatesrdquoPLoSONE vol 10 no 7 Article ID e0131355 2015

[30] I Dıaz-Laviada and N Rodrıguez-Henche ldquoThe potentialantitumor effects of capsaicinrdquo Progress in Drug Research vol68 pp 181ndash208 2014

[31] J G Devassy I D Nwachukwu and P J H Jones ldquoCurcuminand cancer barriers to obtaining a health claimrdquo NutritionReviews vol 73 no 3 pp 155ndash165 2015

[32] C K Singh M A Ndiaye and N Ahmad ldquoResveratroland cancer challenges for clinical translationrdquo Biochimica etBiophysica ActamdashMolecularBasis of Disease vol 1852 no 6 pp1178ndash1185 2015

[33] A Gajowik and M M Dobrzynska ldquoLycopenemdashantioxidantwith radioprotective and anticancer properties a reviewrdquoRoczniki Panstwowego Zakładu Higieny vol 65 no 4 pp 263ndash271 2014

[34] Y A Larasati D D Pamungkas Putri R Y Utomo AHermawan and E Meiyanto ldquoCombination of cisplatin andcinnamon essential oil inhibits HeLa cells proliferation throughcell cycle arrestrdquo Journal of Applied Pharmaceutical Science vol4 no 12 pp 14ndash19 2014

[35] N Arumuggam N A Bhowmick and H P V Rupasinghe ldquoAreview phytochemicals targeting JAKSTAT signaling and IDOexpression in cancerrdquo Phytotherapy Research vol 29 no 6 pp805ndash817 2015

[36] K Matsumoto Y Akao E Kobayashi et al ldquoInduction ofapoptosis by xanthones from mangosteen in human leukemiacell linesrdquo Journal of Natural Products vol 66 no 8 pp 1124ndash1127 2003

[37] K Matsumoto Y Akao H Yi et al ldquoPreferential target ismitochondria in 120572-mangostin-induced apoptosis in humanleukemia HL60 cellsrdquo Bioorganic and Medicinal Chemistry vol12 no 22 pp 5799ndash5806 2004

[38] J-J Chen Z-J Long D-F Xu et al ldquoInhibition of autophagyaugments the anticancer activity of 120572-mangostin in chronicmyeloid leukemia cellsrdquo Leukemia and Lymphoma vol 55 no3 pp 628ndash638 2014

[39] Y Nakagawa M Iinuma T Naoe Y Nozawa and Y AkaoldquoCharacterizedmechanism of 120572-mangostin-induced cell death


caspase-independent apoptosis with release of endonuclease-G from mitochondria and increased miR-143 expression inhuman colorectal cancerDLD-1 cellsrdquoBioorganic andMedicinalChemistry vol 15 no 16 pp 5620ndash5628 2007

[40] H Kurose M-A Shibata M Iinuma and Y Otsuki ldquoAlter-ations in cell cycle and inductionof apoptotic cell death in breastcancer cells treated with 120572-mangostin extracted from mangos-teen pericarprdquo Journal of Biomedicine and Biotechnology vol2012 Article ID 672428 2012

[41] M Yasukawa H Fujihara H Fujimori et al ldquoSynergetic effectsof PARP inhibitor AZD2281 and cisplatin in oral squamouscell carcinoma in Vitro and in Vivordquo International Journal ofMolecular Sciences vol 17 no 3 p E272 2016

[42] M Yasukawa H Fujihara H Fujimori et al ldquoSynergetic effectsof PARP inhibitor AZD2281 and cisplatin in oral squamouscell carcinoma in vitro and in vivordquo International Journal ofMolecular Sciences vol 17 no 3 2016

[43] Y Mizushina I Kuriyama T Nakahara Y Kawashima andH Yoshida ldquoInhibitory effects of 120572-mangostin on mammalianDNA polymerase topoisomerase and human cancer cell pro-liferationrdquo Food and Chemical Toxicology vol 59 pp 793ndash8002013

[44] K Matsumoto Y Akao K Ohguchi et al ldquoXanthones inducecell-cycle arrest and apoptosis in human colon cancer DLD-1cellsrdquo Bioorganic and Medicinal Chemistry vol 13 no 21 pp6064ndash6069 2005

[45] A F A Aisha KMAbu-Salah Z Ismail andAM S AMajidldquo120572-Mangostin enhances betulinic acid cytotoxicity and inhibitscisplatin cytotoxicity on HCT 116 colorectal carcinoma cellsrdquoMolecules vol 17 no 3 pp 2939ndash2954 2012

[46] J Pedraza-Chaverrı L M Reyes-Fermın E G Nolasco-Amayaet al ldquoROS scavenging capacity and neuroprotective effect of120572-mangostin against 3-nitropropionic acid in cerebellar granuleneuronsrdquo Experimental and Toxicologic Pathology vol 61 no 5pp 491ndash501 2009

[47] V Somasundaram R Nadhan K Hemalatha S Kumar Sengo-dan and P Srinivas ldquoNitric oxide and reactive oxygen speciesclues to target oxidative damage repair defective breast cancersrdquoCritical Reviews in OncologyHematology vol 101 pp 184ndash1922016

[48] B Halliwell ldquoDietary polyphenols good bad or indifferent foryour healthrdquo Cardiovascular Research vol 73 no 2 pp 341ndash347 2007

[49] H N Lee H Y Jang H J Kim et al ldquoAntitumor and apoptosis-inducing effects of 120572-mangostin extracted from the pericarpof the mangosteen fruit (Garcinia mangostana L) in YD-15tonguemucoepidermoid carcinoma cellsrdquo International Journalof MolecularMedicine vol 37 no 4 pp 939ndash948 2016

[50] Q Xu J Ma J Lei et al ldquo120572-Mangostin suppresses theviability and epithelial-mesenchymal transition of pancreaticcancer cells by downregulating the PI3KAkt pathwayrdquo BioMedResearch International vol 2014 Article ID 546353 12 pages2014

[51] A Sato H Fujiwara H Oku K Ishiguro and Y Ohizumildquo120572-mangostin induces Ca2+-ATPase-dependent apoptosis viamitochondrial pathway in PC12 cellsrdquo Journal of Pharmacologi-cal Sciences vol 95 no 1 pp 33ndash40 2004

[52] M Y Ibrahim N M Hashim S Mohan et al ldquo120572-Mangostinfrom Cratoxylum arborescens demonstrates apoptogenesis inMCF-7 with regulation of NF-120581B and Hsp70 protein modu-lation in vitro and tumor reduction in vivordquo Drug DesignDevelopment andTherapy vol 8 pp 1629ndash1647 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


the natural compounds from the mangosteen tree on theantiproliferative and secondary effects produced by CDDPHence we decide to conduct such a study with 120572-M becauseit is known to improve the antiproliferative effect of CDDPwhile minimizing its secondary effects [9ndash11] At the sametime it minimally alters normal cells [12ndash14]

Previously we demonstrated that 120572-M has a renopro-tective effect on CDDP-induced nephrotoxicity This effectwas mediated by preventing an increase in ROS tumornecrosis factor-120572 and transforming growth factor 120573 withoutshowing changes in the pharmacokinetics of CDDP [15]Since 120572-M has antiproliferative and renoprotective effectsthe aim of the present study was to evaluate the possibilitythat the combination of this compound with CDDP couldimprove cytotoxicity and diminish secondary effects Thuswe determined the cytotoxic effect of CDDP+120572-M in vitro aswell as in an in vivo cervical cancer model

2 Material and Methods

21Materials 120572-Mwas purified as previously described frommangosteen pericarp [16] purchased from DNP Interna-tional Inc (Whittier CA USA) CDDP 3-(35-dimethylthi-azol-2-yl)25-diphenyltetrazolium (MTT) and 5-(and-6)-carboxy-2101584071015840-dichlorodihydrofluorescein diacetate (car-boxy-H2DCFDA) were obtained from Sigma-Aldrich Co(St Louis MO USA) Dimethyl sulfoxide (DMSO) was ob-tained from MP Biomedicals (Solon OH USA) All rea-gents for flow cytometry were obtained from Millipore Inc(Darmstadt DE USA) All other reagents usedwere obtainedfrom commercial sources

22 Cell Culture Thehuman cervix adenocarcinoma cell line(HeLa)was obtained fromAmericanTypeCulture Collection(Manassas VA USA) Cells were routinely maintained inDulbeccorsquos modified Eaglersquos medium (DMEM) supplementedwith 10 fetal bovine serum (Gibco BRL) and incubated at37∘C under an atmosphere of 5 CO2 at high humidity

23 Cell Viability Assay Mitochondrial function was esti-mated by the MTT assay which is based on the reduction of atetrazolium salt by the mitochondrial dehydrogenase enzymein viable cells After treatment the medium was removed5mgmL MTT was diluted in DMEM without phenol red(1 9) and 100120583L of reagent was added and then the solutionwas incubated for 1 h Formazan dye was dissolved with 2-propanol acid and optical density was measured using anELISA reader at 120582 570nm The results are expressed as thepercentage of MTT reduction in relation to control values

24 Effect of 120572-M and CDDP on Cell Viability The effect of120572-M and CDDP on cell viability was determined using theMTT assay After seeding 1times104 cellswell in culture mediumin 96-well plates 120572-M (0 to 80 120583M) or CDDP (0 to 120 120583M)was added cells were incubated for 24 h and finally cellviability was quantified The percentage of growth inhibitionwas calculated and the concentration was determined atwhich each drug achieved 50 growth inhibition (IC50) Atheoretical isobologram was then constructed to determine

the concentration that would be used for the drug com-binations subsequently evaluated in two different schemescoincubation and preincubation Whereas in the formerscheme both agents were coincubated for 24 h in the latterthe cells were exposed to 120572-M for 24 h and then incubatedwith CDDP for an additional 24 h The results are expressedas the percentage of MTT reduction The experiment wasconducted in triplicate in independent experiments

25 Isobologram The isobologram method was used todetermine the inhibition of cell viability in function of theinteraction between the two drugs [17 18]The IC50 of CDDPalone was plotted on the abscissa and of 120572-M on the ordinateThe combination of these two compounds having additiveeffects should fall on a straight line connecting these twopoints While points above the line represent antagonismthose below the line show synergism

26 Drug Interaction Analysis The combination index (CI)calculated for data analysis of combined drug treatmentwas determined by median-effect principle derived by Chou[19 20] The equation correlates the drug dose and cytotoxiceffect in the following way CI = (11986311198631199091) + (11986321198631199092) +120572(119863111986321198631199091 lowast1198631199092) where1198631 and1198632 represent the concen-trations used in the combined treatment while 1198631199091 and 1198631199092are single treatment concentrations giving the same responseas 1198631 and 1198632 respectively The factor 120572 indicates the typeof interaction 120572 = 0 for similar mechanisms of actionand 120572 = 1 for independent modes of action (120572 = 1 washerein employed) CI = 1 indicates an additive effect CI lt1 synergism and CI gt 1 antagonism

27 Determination of Reactive Oxygen Species (ROS) Pro-duction by Flow Cytometry Cells (2 times 105) were seededin 6-well plates and then treated by using the coincuba-tion or preincubation scheme The cells were later washedwith phosphate-buffered saline solution (PBS) and harvestedwith PBS-EDTA followed by centrifuging at 2000 rpm for8 minutes and resuspension in PBS For ROS detectionthey were treated with 10120583M of the fluorescent marker 5-(and-6)-carboxy-2101584071015840-dichlorodihydrofluorescein diacetate(carboxy-H2DCFDA Sigma-Aldrich) [21] for 30 minutesin the dark Flow cytometry was performed over 10000acquired events with InCyte software (Merck MilliporeDarmstadt DE) with hydrogen peroxide (1mM per 24 h)used as the positive control The experiment was carried outin triplicate

28 Determination of Apoptosis by Flow Cytometry Cells(2 times 105) were seeded in 6-well plates and then treatedby using the coincubation or preincubation scheme Thecells were later washed with PBS and harvested with PBS-EDTA followed by centrifugation at 2000 rpm for 8minutesCells were resuspended in PBS and then treated with GuavaNexin Reagent to measure apoptosis at room temperaturefor 20 minutes in the dark Flow cytometry was performedover 10000 acquired events with Guava Nexin software(Merck Millipore Darmstadt DE) Early and late apoptosiswas determined and the results are expressed as the total









3 Results






CDDP (M)

CDDP

A

Cel

l via

bilit

y (

)

4530150

0

25

50

75

100

125



(a)

A

Cel

l via

bilit

y (

)

CDDP (M)4530150

0

25

50

75

100

125



(b)










Plot P03 ungated

Control

0

40

80Cou

nt 120

180

101 102 103 104 105100


Plot P03 ungated

CDDP

0

40

Cou

nt

80

120

180

101 102 103 104 105100


Plot P03 ungated

-M 10M

0

40

80Cou

nt 120

180

101 102 103 104 105100


-M 15M

Plot P03 ungated

101 102 103 104 105100


0

40

80

120

Cou

nt

180210

Plot P03 ungated

101 102 103 104 105100


0

40

Cou

nt

80

120

180215

Plot P03 ungated

101 102 103 104 105100


0

40

80Cou

nt 120

180

CDDP+-M

Coincubation

(a)

Control2M

CDDP10M-M

15M-M 210 215

00

05

10

15

20

25

Relat

ive R

OS

prod

uctio

n

CDDP+-M

(b)



4 Discussion







Control CDDP -M 10M

-M 15M


210 215

0

40

80Cou

nt 120

180

0

40

80Cou

nt 120

180

0

40

80Cou

nt 120

180

0

40

80

120

Cou

nt

180

0

40

Cou

nt

80

120

180

0

40

80Cou

nt 120

180

101 102 103 104 105100




101 102 103 104 105100




Preincubation

CDDP+-M(a)

Control2M

CDDP10M-M

15M-M 210 215

A B C D

00

05

10

15

20

25

Relat

ive R

OS

prod

uctio

n

CDDP+-M

(b)







Apoptosis


Control

10e0

10e1

10e2

10e3

10e4Apoptosis


10e0

10e1

10e2

10e3

10e4

CDDP

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

-M 10M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

-M 15M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

210

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

215

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

CDDP+-M

Coincubation

(a)

Apop

tosis

()

0

10

20

30

40

Control2M

CDDP10M-M

15M-M 210 215

CDDP+-M

(b)






Apoptosis


Control

Apoptosis


CDDP

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

-M 10M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

215

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

-M 15M 210

CDDP+-M

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

Preincubation

(a)

A

A B C

Apop

tosis

()

210 215Control2M

CDDP10M-M

15M-M

0

20

40

60

CDDP+-M

(b)

































A B C

Control

2 4 6 8 100Time (weeks)

A


Tum

or v

olum

e (m

m3)

times102

0

5

10

15

20

CDDP+-M

(a)

ControlCDDP

-M

0

10

20

30

40

50

Body

wei

ght (

g)

2 4 6 8 100Time (weeks)

CDDP+-M

(b)


A

Control CDDP -M0

1

2

3

4

5

CDDP+-M

Urin

ary

volu

me (

mL

24

h)

(a)Control CDDP -M

0

20

40

60

80

CDDP+-M

A B C

Urin

ary

prot

ein

(mg24

h)

(b)

A B C

Control CDDP -M0

500

1000

1500

2000

CDDP+-M

KIM

-1(p

gm

L)

(c)










Additional Points


Competing Interests


Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results






CDDP (M)

CDDP

A

Cel

l via

bilit

y (

)

4530150

0

25

50

75

100

125



(a)

A

Cel

l via

bilit

y (

)

CDDP (M)4530150

0

25

50

75

100

125



(b)










Plot P03 ungated

Control

0

40

80Cou

nt 120

180

101 102 103 104 105100


Plot P03 ungated

CDDP

0

40

Cou

nt

80

120

180

101 102 103 104 105100


Plot P03 ungated

-M 10M

0

40

80Cou

nt 120

180

101 102 103 104 105100


-M 15M

Plot P03 ungated

101 102 103 104 105100


0

40

80

120

Cou

nt

180210

Plot P03 ungated

101 102 103 104 105100


0

40

Cou

nt

80

120

180215

Plot P03 ungated

101 102 103 104 105100


0

40

80Cou

nt 120

180

CDDP+-M

Coincubation

(a)

Control2M

CDDP10M-M

15M-M 210 215

00

05

10

15

20

25

Relat

ive R

OS

prod

uctio

n

CDDP+-M

(b)



4 Discussion







Control CDDP -M 10M

-M 15M


210 215

0

40

80Cou

nt 120

180

0

40

80Cou

nt 120

180

0

40

80Cou

nt 120

180

0

40

80

120

Cou

nt

180

0

40

Cou

nt

80

120

180

0

40

80Cou

nt 120

180

101 102 103 104 105100




101 102 103 104 105100




Preincubation

CDDP+-M(a)

Control2M

CDDP10M-M

15M-M 210 215

A B C D

00

05

10

15

20

25

Relat

ive R

OS

prod

uctio

n

CDDP+-M

(b)







Apoptosis


Control

10e0

10e1

10e2

10e3

10e4Apoptosis


10e0

10e1

10e2

10e3

10e4

CDDP

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

-M 10M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

-M 15M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

210

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

215

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

CDDP+-M

Coincubation

(a)

Apop

tosis

()

0

10

20

30

40

Control2M

CDDP10M-M

15M-M 210 215

CDDP+-M

(b)






Apoptosis


Control

Apoptosis


CDDP

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

-M 10M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

215

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

-M 15M 210

CDDP+-M

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

Preincubation

(a)

A

A B C

Apop

tosis

()

210 215Control2M

CDDP10M-M

15M-M

0

20

40

60

CDDP+-M

(b)

































A B C

Control

2 4 6 8 100Time (weeks)

A


Tum

or v

olum

e (m

m3)

times102

0

5

10

15

20

CDDP+-M

(a)

ControlCDDP

-M

0

10

20

30

40

50

Body

wei

ght (

g)

2 4 6 8 100Time (weeks)

CDDP+-M

(b)


A

Control CDDP -M0

1

2

3

4

5

CDDP+-M

Urin

ary

volu

me (

mL

24

h)

(a)Control CDDP -M

0

20

40

60

80

CDDP+-M

A B C

Urin

ary

prot

ein

(mg24

h)

(b)

A B C

Control CDDP -M0

500

1000

1500

2000

CDDP+-M

KIM

-1(p

gm

L)

(c)










Additional Points


Competing Interests


Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CDDP (M)

CDDP

A

Cel

l via

bilit

y (

)

4530150

0

25

50

75

100

125



(a)

A

Cel

l via

bilit

y (

)

CDDP (M)4530150

0

25

50

75

100

125



(b)










Plot P03 ungated

Control

0

40

80Cou

nt 120

180

101 102 103 104 105100


Plot P03 ungated

CDDP

0

40

Cou

nt

80

120

180

101 102 103 104 105100


Plot P03 ungated

-M 10M

0

40

80Cou

nt 120

180

101 102 103 104 105100


-M 15M

Plot P03 ungated

101 102 103 104 105100


0

40

80

120

Cou

nt

180210

Plot P03 ungated

101 102 103 104 105100


0

40

Cou

nt

80

120

180215

Plot P03 ungated

101 102 103 104 105100


0

40

80Cou

nt 120

180

CDDP+-M

Coincubation

(a)

Control2M

CDDP10M-M

15M-M 210 215

00

05

10

15

20

25

Relat

ive R

OS

prod

uctio

n

CDDP+-M

(b)



4 Discussion







Control CDDP -M 10M

-M 15M


210 215

0

40

80Cou

nt 120

180

0

40

80Cou

nt 120

180

0

40

80Cou

nt 120

180

0

40

80

120

Cou

nt

180

0

40

Cou

nt

80

120

180

0

40

80Cou

nt 120

180

101 102 103 104 105100




101 102 103 104 105100




Preincubation

CDDP+-M(a)

Control2M

CDDP10M-M

15M-M 210 215

A B C D

00

05

10

15

20

25

Relat

ive R

OS

prod

uctio

n

CDDP+-M

(b)







Apoptosis


Control

10e0

10e1

10e2

10e3

10e4Apoptosis


10e0

10e1

10e2

10e3

10e4

CDDP

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

-M 10M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

-M 15M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

210

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

215

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

CDDP+-M

Coincubation

(a)

Apop

tosis

()

0

10

20

30

40

Control2M

CDDP10M-M

15M-M 210 215

CDDP+-M

(b)






Apoptosis


Control

Apoptosis


CDDP

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

-M 10M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

215

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

-M 15M 210

CDDP+-M

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

Preincubation

(a)

A

A B C

Apop

tosis

()

210 215Control2M

CDDP10M-M

15M-M

0

20

40

60

CDDP+-M

(b)

































A B C

Control

2 4 6 8 100Time (weeks)

A


Tum

or v

olum

e (m

m3)

times102

0

5

10

15

20

CDDP+-M

(a)

ControlCDDP

-M

0

10

20

30

40

50

Body

wei

ght (

g)

2 4 6 8 100Time (weeks)

CDDP+-M

(b)


A

Control CDDP -M0

1

2

3

4

5

CDDP+-M

Urin

ary

volu

me (

mL

24

h)

(a)Control CDDP -M

0

20

40

60

80

CDDP+-M

A B C

Urin

ary

prot

ein

(mg24

h)

(b)

A B C

Control CDDP -M0

500

1000

1500

2000

CDDP+-M

KIM

-1(p

gm

L)

(c)










Additional Points


Competing Interests


Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Plot P03 ungated

Control

0

40

80Cou

nt 120

180

101 102 103 104 105100


Plot P03 ungated

CDDP

0

40

Cou

nt

80

120

180

101 102 103 104 105100


Plot P03 ungated

-M 10M

0

40

80Cou

nt 120

180

101 102 103 104 105100


-M 15M

Plot P03 ungated

101 102 103 104 105100


0

40

80

120

Cou

nt

180210

Plot P03 ungated

101 102 103 104 105100


0

40

Cou

nt

80

120

180215

Plot P03 ungated

101 102 103 104 105100


0

40

80Cou

nt 120

180

CDDP+-M

Coincubation

(a)

Control2M

CDDP10M-M

15M-M 210 215

00

05

10

15

20

25

Relat

ive R

OS

prod

uctio

n

CDDP+-M

(b)



4 Discussion







Control CDDP -M 10M

-M 15M


210 215

0

40

80Cou

nt 120

180

0

40

80Cou

nt 120

180

0

40

80Cou

nt 120

180

0

40

80

120

Cou

nt

180

0

40

Cou

nt

80

120

180

0

40

80Cou

nt 120

180

101 102 103 104 105100




101 102 103 104 105100




Preincubation

CDDP+-M(a)

Control2M

CDDP10M-M

15M-M 210 215

A B C D

00

05

10

15

20

25

Relat

ive R

OS

prod

uctio

n

CDDP+-M

(b)







Apoptosis


Control

10e0

10e1

10e2

10e3

10e4Apoptosis


10e0

10e1

10e2

10e3

10e4

CDDP

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

-M 10M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

-M 15M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

210

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

215

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

CDDP+-M

Coincubation

(a)

Apop

tosis

()

0

10

20

30

40

Control2M

CDDP10M-M

15M-M 210 215

CDDP+-M

(b)






Apoptosis


Control

Apoptosis


CDDP

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

-M 10M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

215

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

-M 15M 210

CDDP+-M

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

Preincubation

(a)

A

A B C

Apop

tosis

()

210 215Control2M

CDDP10M-M

15M-M

0

20

40

60

CDDP+-M

(b)

































A B C

Control

2 4 6 8 100Time (weeks)

A


Tum

or v

olum

e (m

m3)

times102

0

5

10

15

20

CDDP+-M

(a)

ControlCDDP

-M

0

10

20

30

40

50

Body

wei

ght (

g)

2 4 6 8 100Time (weeks)

CDDP+-M

(b)


A

Control CDDP -M0

1

2

3

4

5

CDDP+-M

Urin

ary

volu

me (

mL

24

h)

(a)Control CDDP -M

0

20

40

60

80

CDDP+-M

A B C

Urin

ary

prot

ein

(mg24

h)

(b)

A B C

Control CDDP -M0

500

1000

1500

2000

CDDP+-M

KIM

-1(p

gm

L)

(c)










Additional Points


Competing Interests


Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Control CDDP -M 10M

-M 15M


210 215

0

40

80Cou

nt 120

180

0

40

80Cou

nt 120

180

0

40

80Cou

nt 120

180

0

40

80

120

Cou

nt

180

0

40

Cou

nt

80

120

180

0

40

80Cou

nt 120

180

101 102 103 104 105100




101 102 103 104 105100




Preincubation

CDDP+-M(a)

Control2M

CDDP10M-M

15M-M 210 215

A B C D

00

05

10

15

20

25

Relat

ive R

OS

prod

uctio

n

CDDP+-M

(b)







Apoptosis


Control

10e0

10e1

10e2

10e3

10e4Apoptosis


10e0

10e1

10e2

10e3

10e4

CDDP

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

-M 10M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

-M 15M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

210

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

215

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

CDDP+-M

Coincubation

(a)

Apop

tosis

()

0

10

20

30

40

Control2M

CDDP10M-M

15M-M 210 215

CDDP+-M

(b)






Apoptosis


Control

Apoptosis


CDDP

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

-M 10M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

215

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

-M 15M 210

CDDP+-M

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

Preincubation

(a)

A

A B C

Apop

tosis

()

210 215Control2M

CDDP10M-M

15M-M

0

20

40

60

CDDP+-M

(b)

































A B C

Control

2 4 6 8 100Time (weeks)

A


Tum

or v

olum

e (m

m3)

times102

0

5

10

15

20

CDDP+-M

(a)

ControlCDDP

-M

0

10

20

30

40

50

Body

wei

ght (

g)

2 4 6 8 100Time (weeks)

CDDP+-M

(b)


A

Control CDDP -M0

1

2

3

4

5

CDDP+-M

Urin

ary

volu

me (

mL

24

h)

(a)Control CDDP -M

0

20

40

60

80

CDDP+-M

A B C

Urin

ary

prot

ein

(mg24

h)

(b)

A B C

Control CDDP -M0

500

1000

1500

2000

CDDP+-M

KIM

-1(p

gm

L)

(c)










Additional Points


Competing Interests


Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Apoptosis


Control

10e0

10e1

10e2

10e3

10e4Apoptosis


10e0

10e1

10e2

10e3

10e4

CDDP

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

-M 10M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

-M 15M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

210

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

10e0

10e1

10e2

10e3

10e4

215

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

CDDP+-M

Coincubation

(a)

Apop

tosis

()

0

10

20

30

40

Control2M

CDDP10M-M

15M-M 210 215

CDDP+-M

(b)






Apoptosis


Control

Apoptosis


CDDP

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

-M 10M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

215

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

-M 15M 210

CDDP+-M

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

Preincubation

(a)

A

A B C

Apop

tosis

()

210 215Control2M

CDDP10M-M

15M-M

0

20

40

60

CDDP+-M

(b)

































A B C

Control

2 4 6 8 100Time (weeks)

A


Tum

or v

olum

e (m

m3)

times102

0

5

10

15

20

CDDP+-M

(a)

ControlCDDP

-M

0

10

20

30

40

50

Body

wei

ght (

g)

2 4 6 8 100Time (weeks)

CDDP+-M

(b)


A

Control CDDP -M0

1

2

3

4

5

CDDP+-M

Urin

ary

volu

me (

mL

24

h)

(a)Control CDDP -M

0

20

40

60

80

CDDP+-M

A B C

Urin

ary

prot

ein

(mg24

h)

(b)

A B C

Control CDDP -M0

500

1000

1500

2000

CDDP+-M

KIM

-1(p

gm

L)

(c)










Additional Points


Competing Interests


Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Apoptosis


Control

Apoptosis


CDDP

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

-M 10M

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

Annexin V-PE (PM1)

Apoptosis

10e410e310e210e110e0

215

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

10e0

10e1

10e2

10e3

10e4

-M 15M 210

CDDP+-M

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

7-A

AD

(PM

2)

Preincubation

(a)

A

A B C

Apop

tosis

()

210 215Control2M

CDDP10M-M

15M-M

0

20

40

60

CDDP+-M

(b)

































A B C

Control

2 4 6 8 100Time (weeks)

A


Tum

or v

olum

e (m

m3)

times102

0

5

10

15

20

CDDP+-M

(a)

ControlCDDP

-M

0

10

20

30

40

50

Body

wei

ght (

g)

2 4 6 8 100Time (weeks)

CDDP+-M

(b)


A

Control CDDP -M0

1

2

3

4

5

CDDP+-M

Urin

ary

volu

me (

mL

24

h)

(a)Control CDDP -M

0

20

40

60

80

CDDP+-M

A B C

Urin

ary

prot

ein

(mg24

h)

(b)

A B C

Control CDDP -M0

500

1000

1500

2000

CDDP+-M

KIM

-1(p

gm

L)

(c)










Additional Points


Competing Interests


Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































A B C

Control

2 4 6 8 100Time (weeks)

A


Tum

or v

olum

e (m

m3)

times102

0

5

10

15

20

CDDP+-M

(a)

ControlCDDP

-M

0

10

20

30

40

50

Body

wei

ght (

g)

2 4 6 8 100Time (weeks)

CDDP+-M

(b)


A

Control CDDP -M0

1

2

3

4

5

CDDP+-M

Urin

ary

volu

me (

mL

24

h)

(a)Control CDDP -M

0

20

40

60

80

CDDP+-M

A B C

Urin

ary

prot

ein

(mg24

h)

(b)

A B C

Control CDDP -M0

500

1000

1500

2000

CDDP+-M

KIM

-1(p

gm

L)

(c)










Additional Points


Competing Interests


Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















A B C

Control

2 4 6 8 100Time (weeks)

A


Tum

or v

olum

e (m

m3)

times102

0

5

10

15

20

CDDP+-M

(a)

ControlCDDP

-M

0

10

20

30

40

50

Body

wei

ght (

g)

2 4 6 8 100Time (weeks)

CDDP+-M

(b)


A

Control CDDP -M0

1

2

3

4

5

CDDP+-M

Urin

ary

volu

me (

mL

24

h)

(a)Control CDDP -M

0

20

40

60

80

CDDP+-M

A B C

Urin

ary

prot

ein

(mg24

h)

(b)

A B C

Control CDDP -M0

500

1000

1500

2000

CDDP+-M

KIM

-1(p

gm

L)

(c)










Additional Points


Competing Interests


Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























Additional Points


Competing Interests


Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Synergic Effect of -Mangostin on the ...downloads.hindawi.com/journals/omcl/2016/7981397.pdfect against CDDP-induced nephrotoxicity, as well as antioxidant, antitumor,