Vitamin C and the Red Wine Polyphenol Resveratrol - but ... · asterisk (*), a P-value

Article ID: WMC004329 ISSN 2046-1690

Vitamin C and the Red Wine Polyphenol Resveratrol- but not Curcumin and the Glycolysis Inhibitors2-Deoxyglucose, Dichloroacetate and3-Bromopyruvate - Induce Selective Cytotoxicityagainst Lung Cancer CellsCorresponding Author:Dr. Miguel Lopez-Lazaro,Associate Professor, Department of Pharmacology, Faculty of Pharmacy, University of Seville - Spain

Submitting Author:Dr. Miguel Lopez-Lazaro,Associate Professor, Department of Pharmacology, Faculty of Pharmacy, University of Seville - Spain

Other Authors:Dr. José Manuel Calderón-Montaño,Postdoctoral Researcher, Department of Pharmacology, Faculty of Pharmacy, University of Seville - Spain

Ms. Estefanía Burgos-Morón,PhD Student, Department of Pharmacology, Faculty of Pharmacy, University of Seville - Spain

Ms. Sara María Martínez-Sánchez,PhD Student, Department of Pharmacology, Faculty of Pharmacy, University of Seville - Spain

Article ID: WMC004329

Article Type: Research articles

Submitted on:11-Jul-2013, 03:56:11 PM GMT Published on: 12-Jul-2013, 06:30:28 AM GMT

Article URL: http://www.webmedcentral.com/article_view/4329

Subject Categories:CANCER

Keywords:anticancer, tumor, antitumor, etoposide, 5-fluorouracil, oxaliplatin, cisplatin, read grape.

How to cite the article:Calderón-Montaño J, Burgos-Morón E, Martínez-Sánchez S, Lopez-Lazaro M. Vitamin Cand the Red Wine Polyphenol Resveratrol - but not Curcumin and the Glycolysis Inhibitors 2-Deoxyglucose,Dichloroacetate and 3-Bromopyruvate - Induce Selective Cytotoxicity against Lung Cancer Cells.WebmedCentral CANCER 2013;4(7):WMC004329

Copyright: This is an open-access article distributed under the terms of the Creative Commons AttributionLicense(CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided theoriginal author and source are credited.

Source(s) of Funding:

University of Seville.

Competing Interests:

The authors declare that they do not have conflicts of interest.

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Vitamin C and the Red Wine Polyphenol Resveratrol- but not Curcumin and the Glycolysis Inhibitors2-Deoxyglucose, Dichloroacetate and3-Bromopyruvate - Induce Selective Cytotoxicityagainst Lung Cancer CellsAuthor(s): Calderón-Montaño J, Burgos-Morón E, Martínez-Sánchez S, Lopez-Lazaro M

Abstract

Cancer statistics show that the most commonlydiagnosed cancer in the world is lung cancer, that over50% of patients diagnosed with this cancer havedistant metastasis, and that only 4% of these patientsmanage to survive more than 5 years. The limitedselective cytotoxicity of the drugs used for thetreatment of these patients probably accounts forthese high mortality rates. In this work, we haveassessed the selective anticancer activity of severaldrugs currently undergoing clinical trials by usinghuman A549 lung cancer cells and human MRC5non-malignant lung fibroblasts. Vitamin C and the redwine polyphenol resveratrol induced selectivecytotoxicity towards the cancer cell line. Vitamin C (1mM) induced higher selective cytotoxicity than theanticancer agents cisplatin, oxaliplatin, etoposide and5-fluorouracil. A lyophilized red wine extract, but not ahydroalcoholic extract from red grapes, also showedcertain selectivity against lung cancer cells. Neitherthe curry polyphenol curcumin nor the glycolysisinhibitors 2-deoxyglucose, dichloroacetate and3-bromopyruvate displayed selective cytotoxicity. Wealso report that A549 lung cancer cells have higherglycolytic rates (higher glucose consumption andhigher lactate production) than human MRC5non-malignant lung fibroblasts, and that thecombination of each glycolytic inhibitor with thepro-oxidant agents pyrogallol and hydrogen peroxidedoes not result in a significant increase in theircytotoxicity or selectivity against the cancer cell line.Our results support the possible evaluation of vitaminC and resveratrol in clinical trial for the treatment ofmetastatic lung cancers, and suggest that curcuminand the glycolysis inhibitors 2-deoxyglucose,dichloroacetate and 3-bromopyruvate have a limitedpotential (at least as single agents) for the treatment ofpatients with this type of cancer.

Introduction

Global cancer statistics show that lung cancer is themost common cancer and the leading cause of cancerdeath [1]. The latest global cancer statistics show that,in 2008, lung cancer was the most commonlydiagnosed cancer and the leading cause of cancerdeath in males. Among females, lung cancer was thefourth most commonly diagnosed cancer and thesecond leading cause of cancer death. This type ofcancer accounts for 13% (1.6 million) of the totalcases and 18% (1.4 million) of the deaths in 2008 [1].In the United States, the American Cancer Societyestimates 228,190 new lung cancer cases and159,480 lung cancer deaths for the year 2013 [2].Perhaps the most worrying data are those showingthat over 50% of patients diagnosed with lung cancerhave distant metastasis, and that only 4% of thesepatients manage to survive more than 5 years [2].Many cancer cells in patients with metastatic lungcancers are not localized and, therefore, cannot beeliminated by surgery or radiation therapy. Thesepatients need to be treated systemically withanticancer drugs. Although these drugs can kill lungcancer cells, most of them are also toxic tonon-malignant cells, cause severe side effects inpatients and, therefore, need to be used at suboptimalconcentrations. The low selective cytotoxicity of thedrugs used for the treatment patients with metastaticlung cancers probably accounts for the high mortalityrates observed in these patients.

Several natural products (e.g., vitamin C [3-7],resveratrol [8-12] and curcumin [13-17]) and severalsynthetic glycolytic inhibitors (e.g., 2-deoxyglucose,dichloroacetate and 3-bromopyruvate) [18-24] haveshown promising anticancer effects in preclinicalmodels. Indeed, vitamin C, resveratrol, curcumin,2-deoxyglucose, and dichloroacetate have enteredclinical trials for the treatment of specific cancers (seehttp://clinicaltrials.gov/). By using human A549 lung



cancer cells and human MRC5 non-malignant lungcells, here we have evaluated the selective anticanceractivity of these drugs with the aim of identifyingpotential new treatments for patients with metastaticlung cancers.

Material and Methods

Chemicals and cell lines

Ascorbic acid (vitamin C; 99%), resveratrol (99%),curcumin (70%), 2-deoxyglucose (2-deoxy-D-glucose;98%), dichloroacetate (98%), 3-bromopyruvate (97%),etoposide (98%), 5-fluorouracil (99%), oxaliplatin (99%)and cisplatin (99.9%) were purchased from Sigma.Their chemical structures are represented inIllustration 1. The human A549 lung cancer cell lineand the human embryo lung fibroblastic MRC-5 cellline were purchased from the European Collection ofCell Cultures. Both cell lines were maintained inDMEM supplemented with 2 mM glutamine, 50 µg/mLpenicillin, 50 µg/mL streptomycin and 10% fetal bovineserum, and were cultured at 37°C in a humidifiedatmosphere containing 5% CO2. Cell culture reagentswere obtained from Life Technologies.

Preparation of the extracts

Commercial red grapes were extracted withethanol:water (1:1) at 60°C for 1 hour by using anultrasound water bath apparatus. Ethanol was theneliminated in a rotary vacuum evaporator and theremaining water solution was lyophilized. Commercialred wine was directly lyophilized, obtaining an alcoholfree-lyophilized red wine extract.

Assay for cytotoxic activity (MTT assay)

The MTT assay is a colorimetric technique that allowsthe quantitative determination of cell viability. It isbased on the capability of viable cells to transform theMTT sa l t (3 - (4 ,5 -d imethy l th iazo l -2 -y l ) -2,5-diphenyltetrazolium bromide) into a formazan dye.Exponentially growing cells were seeded into 96-wellplates and drugs were added 24 h later. Following anincubation period specified in figure legends (generally48 h), medium was removed and 125 µL MTT (1mg/mL in medium) was added to each well for 4 hours.Then, 80 µL 20% SDS in 0.02 M HCl were added,plates were incubated for 10 hours at 37 ºC, andoptical densities were measured at 540 nm on amultiwell plate spectrophotometer reader. Cell viabilitywas expressed as percentage in relation to controls.All data were averaged from at least threeindependent experiments and were expressed asmeans ± standard error of the means (SEM).

Measurement of glycolytic rates

Glycolysis rates were assessed by measuringconcentrations of glucose (initial product of glycolysis)and lactate (final product of glycolysis) in supernatantsof A549 lung cancer cells and MRC-5 lungnon-malignant cells. Briefly, 4 x 105 cells were allowedto grow in 24-well plates for 8 h. After medium removal,cells were washed with PBS and 300 µL of freshmedium were added to each well. Afterwards, cellswere allowed to grow for 8 h, and glucose and lactateconcentrations were determined in cell supernatantsby using the Accutrend® Plus analyzer together withAccutrend glucose strips and BM-Lactate Strips(Roche Diagnostics). After calibrating the instrumentwith glucose and lactate calibration strips, test stripswere used to determine glucose and lactate levels viacolorimetric-oxidase mediator reactions according tothe manufacturer's instructions [25]. Results areshown as means ± standard error of the means (SEM)of three independent experiments.

Inhibition of glycolysis

Glycolysis inhibition was assessed by measuringconcentrations of glucose and lactate in control andtreated cells. Briefly, 4 x 105 cells were allowed togrow in 24-well plates for 8 h. After medium removal,cells were washed with PBS and 300 µL of freshmedium were added to each well. Afterwards, drugswere added and, after an incubation period 8 h,glucose and lactate concentrations were determined incell supernatants as described in the previous section.Results are expressed as percentage of lactateproduction and percentage of glucose consumption inrelation to untreated cells, and are shown as means ±standard error of the means (SEM) of threeindependent experiments.

Statistical analysis

All data were averaged from at least threeindependent experiments and were expressed asmeans ± standard error of the means (SEM). Forstatistical analysis we used the t-test (paired,two-tailed). A P-value >0.05 is not consideredstatistically significant and is not represented by anysymbol. A P-value <0.05 is considered to correspondwith statistical significance and is indicated with anasterisk (*), a P-value <0.01 is indicated with a doubleasterisk (**), and a P-value <0.001 is indicated with atriple asterisk (***).

Results and Discussion

Most patients with metastatic lung cancers diebecause the drugs used for the treatment of their



disease have a limited capacity to selectively kill lungcancer cells. The initial aim of this work was theevaluation of the selective anticancer activity ofseveral compounds that have shown anticancerpotential (and that have entered clinical trials), with thehope of identifying potential new treatments for thesepatients. We initially selected the natural productsascorbic acid (vitamin C), resveratrol and curcumin(Illustration 1), and their possible selective cytotoxicactivity was evaluated by using the human A549 lungcancer cell l ine and the human embryo lungfibroblastic MRC-5 cell line. These malignant andnon-malignant cells were exposed for 48 h to thesethree natural products and to the commonly usedanticancer agents etoposide, cisplatin and oxaliplatin;then cell viability was estimated with the MTT assay.Results, represented in Illustration 2, show that vitaminC and the red wine constituent resveratrol induced astatistically significant selective cytotoxicity towardsthe cancer cell line. It is worth noting that theselectivity cytotoxicity shown by ascorbic acid at aconcentration of 1 mM was higher than that observedfor any concentration of the three tested anticanceragents. We also prepared and evaluated the selectivecytotoxicity of a lyophilized red wine extract and of ahydroalcoholic extract from commercial red grapes.Illustration 3 shows that the alcohol free-lyophilizedred wine extract, but not the hydroalcoholic extractform red grapes, had certain selectivity towards thelung cancer cell line. We cannot conclude, however,that resveratrol is responsible for this activity, as redwine contains other polyphenols that may play a rolein its selective cytotoxicity [26].

The curry polyphenol curcumin, which have enteredclinical trials for the treatment of several cancers, didnot show selective cytotoxicity towards the lung cancercell line (Illustration 2). Although this dietaryconstituent has anticancer potential [13;14], it is theauthors’ opinion that its chemotherapeutic potentialmay have been overdiscussed in the last years [15;27].

Our next goal was to evaluate the selective anticanceractivity of the glycolysis inhibitors 2-deoxyglucose,dichloroacetate and 3-bromopyruvate. Evidencesuggests that cancer cells keep sustained glycolyticrates despite the presence of an adequate oxygensupply (Warburg Effect), and that these high glycolyticrates play a key role in their survival. The inhibition ofglycolysis has become in recent years an attractivestrategy to selectively kill cancer cells [28-32]. Theglycolytic inhibitors 2-deoxyglucose, dichloroacetateand 3-bromopyruvate have shown promisinganticancer effects in preclinical models [18-24] and2-deoxyglucose and dichloroacetate have already

entered clinical trials for the treatment of specificcancers (http://clinicaltrials.gov/). Results representedin Illustration 4 show, however, that none of thesethree glycolytic inhibitors displayed selectivecytotoxicity against the cancer cell line. In fact,non-malignant lung fibroblasts were somewhat moresensitive to their cytotoxic activity than lung cancercells.

With the aim of understanding these unexpectedresults, we considered the possibility that the cancercell line did not have increased glycolytic rates inrelation to the non-malignant cell line. We alsospeculated with the possibility that these glycolysisinhibitors were not inhibiting glycolysis in these cells.Results represented in Illustration 5 A show, however,that the A549 lung cancer cells consumed moreglucose and produced more lactate than the MRC5non-malignant lung cells. Since glucose and lactaterespectively are the initial and final products ofglycolysis, our data indicate that the cancer cell linehave higher glycolytic rates than the non-malignantcell line. Results represented in Illustration 5 B showthat dichloroacetate inhibited glucose consumptionand lactate production in both cell lines. Therefore, thelack of selective cytotoxicity displayed by the glycolyticinhibitors (Illustration 4) cannot be explained by similarglycolytic rates in both cell lines or by lack of inhibitionof glycolysis in these cell lines. Perhaps these drugsmay induce cytotoxicity against these cells throughglycolysis-independent mechanisms at lowerconcentrations than those required to inhibit glycolysis.

Cancer cells are known to produce high levels ofhydrogen peroxide constitutively [33;34]. We haverecently discussed that the activation of glycolysis incancer cells may play a key role in the detoxification ofhydrogen peroxide by increasing the levels of thehydrogen peroxide scavenger pyruvate and byregenerating NADPH [32]. We also hypothesized thatthe combination of glycolytic inhibitors with pro-oxidantagents might be therapeutically useful [32]. In a recentpaper, Vuyyuri et al. reported that combinations ofv i tamin C w i th the g lyco lys i s i nh ib i to r3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO)synergistically enhanced cell death in lung cancer cellsbut not in non-malignant cells [6]. These data supportour hypothesis [32], as vitamin C is known to inducecancer cell death through generation of hydrogenperoxide [3]. To further support this hypothesis, weevaluated whether the combination of the glycolyticinhibitors 2-deoxyglucose, dichloroacetate and3-bromopyruvate with hydrogen peroxide and thehydrogen peroxide-generating agent pyrogallol [35]resulted in an increase in their cytotoxicity or



selectivity against the cancer cell line. Our resultsrevealed, however, that the combination of theseglycolytic inhibitors with the pro-oxidant agentspyrogallol and hydrogen peroxide did not result in asignificant increase in their cytotoxicity or selectivityagainst the cancer cell line (Illustration 6).

In conclusion, this report shows that vitamin C and thered wine polyphenol resveratrol induce selectivecytotoxicity towards lung cancer cells. Because thesenatural products have already entered clinical trials forthe treatment of specific cancers, these data supporttheir possible advancement into clinical trials for thetreatment of metastatic lung cancers. Although thedietary agent curcumin and the glycolysis inhibitors2-deoxyglucose and dichloroacetate are in clinicaltrials for the treatment of specific cancers, our datasuggest that these agents have a limited potential (atleast as single agents) for the treatment of patientswith lung cancer.

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Illustrations

Illustration 1

Chemical structures of the drugs evaluated in this article



Illustration 2

Selective cytotoxic activity of vitamin C and resveratrol. Human A549 lung cancer cells and human MRC-5 non-malignant lungfibroblasts were exposed for 48 h to vitamin C, resveratrol, curcumin and the anticancer drugs etoposide, cisplatin and oxaliplatin.Cell viability was then estimated with the MTT assay.



Illustration 3

Selective cytotoxic activity of a lyophilized read wine extract. A549 lung cancer cells and MRC-5 non-malignant lung fibroblastswere exposed for 48 h to a lyophilized read wine extract and to a hydroalcoholic extract from commercial read grapes. Cell viabilitywas then estimated with the MTT assay.



Illustration 4

Glycolysis inhibitors 2-deoxyglucose, dichloroacetate and 3-bromopyruvate do not induce selective cytotoxicity towards lung cancercells. Human A549 lung cancer cells and human MRC-5 non-malignant lung fibroblasts were exposed for 48 h to these threeglycolysis inhibitors and to the anticancer drug 5-fluorouracil. Cell viability was estimated with the MTT assay.



Illustration 5

Human A549 lung cancer cells have higher glycolytic rates than human MRC-5 non-malignant lung fibroblasts (A). The glycolysisinhibitor dichloroacetate (DCA) inhibits glycolysis in both cell lines (B). Glycolytic rates and glycolysis inhibition was assessed bymeasuring glucose and lactate levels in cell supernatants (see Material and Methods).



Illustration 6

Combinations of the glycolysis inhibitors 2-deoxyglucose, dichloroacetate and 3-bromopyruvate with the pro-oxidant agentspyrogallol and hydrogen peroxide do not significantly increase their cytotoxicity or selectivity towards the lung cancer cell line. Theglycolysis inhibitor was added to the cells 4 h before the pro-oxidant agent. After a combined exposure of 4 h, cells were washedwith PBS and were grown in fresh medium for 40 h. Then, cell viability was estimated with the MTT assay.



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Vitamin C and the Red Wine Polyphenol Resveratrol - but ... · asterisk (*), a P-value

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