α-Hexylcinnamaldehyde (HCA), a compound derived from cinnamaldehyde, was evaluated for its potential chemosensitizing properties

DI GIACOMO, Silvia

DI SOTTO, Antonella

El Readi, Mahmoud Zaki

Mazzanti, Gabriela

Wink, Michael

English

DI GIACOMO, SILVIA

DI SOTTO, ANTONELLA

MAZZANTI, Gabriela

Archivio della ricerca- Università di Roma La Sapienza

Abstract. Aim: α-Hexylcinnamaldehyde (HCA), a compoundderived from cinnamaldehyde, was evaluated for its potentialchemosensitizing properties. Materials and Methods: Thecytotoxicity of HCA was tested against Caco-2, CCRF/CEMand CEM/ADR5000 human cancer cells. Furthermore, itsability to increase doxorubicin cytotoxicity was evaluated incombination assays. Rhodamine123 efflux assay was carriedout in order to highlight the possible interference of HCAwith functionality of ATP-binding cassette (ABC)-transporters. Results: In spite of a low cytotoxicity, HCAincreased the antiproliferative effect of doxorubicin in all thecell lines tested, being particularly effective in CCRF/CEM.The compound also reduced the rhodamine123 efflux inCaco-2 and CEM/ADR5000 cells, suggesting a possibleinterference with ABC transporter functionality. Conclusion:Considering that the greatest synergism between HCA andDOX was found against CCRF/CEM cells (lacking ABCpumps), it seems likely that non-specific mechanisms,including the alteration of membrane permeability, could beinvolved in the chemosensitizing effect of HCA.Chemotherapy with cytotoxic drugs represents the treatment ofchoice for patients diagnosed with locally advanced andmetastatic cancer. Unfortunately, many oncological patientsoften became insensitive to a variety of structurally andmechanistically related and unrelated antitumor drugs, due tothe development of multidrug resistance (MDR). MDR isdefined as a multifactorial phenomenon in which both cellular(due to the alterations of the malignant cell biochemistry) andnon-cellular mechanisms have been included (1). The first canbe classified into non-classical and transport-based classicalMDR phenotypes. Non-classical MDR mechanisms includeinterference with the activity of some enzymes (e.g. glutathioneS-transferase and topoisomerase) that can reduce chemotherapyefficacy, and changes in the balance of proteins involved inapoptosis. Conversely, transport-based classical MDRmechanisms are related to the ATP-binding cassette (ABC)-transporter function (1), among which P-glycoprotein (MDR1;encoded by ABCB1), multidrug resistance protein (MRP1;encoded by ABCC1), and breast cancer resistance protein(BCRP; encoded by ABCG2) have been most extensivelystudied (1). These proteins are responsible for drug efflux fromcells, so their overexpression in cancer cells can reduceanticancer drug bioavailability and favor MDR development.In this context, inhibiting ABC transporter function (or their expression) by weakly cytotoxic agents (namelychemosensitizers) represents a promising approach for MDR-reversion and chemotherapy success. Furthermore, incombination with a chemosensitizer, lower doses of anticancerdrugs are effective, hence the adverse effects of chemotherapycan be reduced (2). In spite of their promising properties,current MDR-reversal agents (e.g. verapamil) are only weaklyeffective in vivo or produce too severe side-effects; therefore,new bioactive compounds are still needed (3).Cinnamaldehyde (cinnamic aldehyde, or 3-phenyl-2-propenal), the major component (60-75%) of cinnamon bark(Cinnamomum spp., Lauraceae) essential oil, has been foundto possess beneficial properties (including antimutagenic,antioxidative, anti-inflammatory, and antiproliferative) andhas been shown to inhibit the MRP1 transporter (4, 5). Inspite of these activities, the clinical application ofcinnamaldehyde is limited due to its poor chemical stabilityand rapid clearance (half-life of about 4 min) (6). Therefore,some alkyl-derivatives have been developed in order to makethe molecule less reactive and more stable. In the presentstudy, the semisynthetic derivative α-hexylcinnamaldehyde(HCA; Figure 1), widely used as an additive in food,cosmetic and pharmaceutical industries (4), was investigatedfor its chemosensitizing properties in human cancer cells.3347Correspondence to: Antonella Di Sotto, Department of Physiologyand Pharmacology V. Erspamer, Sapienza University of Rome, P.leAldo Moro 5, 00185 Rome, Italy. Tel: +39 0649912904, Fax: +390649912480, e-mail: antonella.disotto@uniroma1.itKey Words: Cinnamaldehyde, naturally-derived compound,chemosensitizer, MDR reversal agent, ABC-transporters.ANTICANCER RESEARCH 36: 3347-3352 (2016)α-Hexylcinnamaldehyde Synergistically Increases DoxorubicinCytotoxicity Towards Human Cancer Cell LinesSILVIA DI GIACOMO1, ANTONELLA DI SOTTO1, MAHMOUD ZAKI EL-READI2, GABRIELA MAZZANTI1 and MICHAEL WINK21Department of Physiology and Pharmacology V. Erspamer, Sapienza University of Rome, Rome, Italy;2Institute of Pharmacy and Molecular Biotechnology, Ruprecht Karls University, Heidelberg, Germany0250-7005/2016 $2.00+.40Materials and MethodsCell lines. Human colorectal adenocarcinoma Caco-2 and T-cellleukemia CCRF/CEM (Leibniz Institute DSMZ-German Collectionof Microorganisms and Cell Cultures, Braunschweig, Germany) andCEM/ADR5000 cells (a MDR1-overexpressing and doxorubicin-resistant leukemia subline kindly provided by Professor ThomasEfferth, German Cancer Research Center, Heidelberg) were usedand cultivated according to El-Readi et al. (7). Cytotoxicity. Cell viability was evaluated by the 3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide (MTT)assay (7) and determined as follow:Viability (%)=[(ODtreated cells−ODmedium control)/(ODuntreated cells−ODmedium control)]×100.Combination assay. Cytotoxicity of doxorubicin (0.00005-1,000μM) was tested, by MTT assay, in the presence of HCA, atinhibitory concentrations (IC) which induce 10% (IC10) and 20%(IC20) cytotoxicity, as found by the dose–response curve. The typeof interaction (synergistic, additive or antagonistic) was evaluatedby combination index (CI), isobologram analysis (IB), median-effect equation (r-value), and reversal ratio value (RR) (4, 8).Rhodamine 123 efflux assay. Rhodamine 123 (Rho123) is afluorescent dye effluxed by P-gp and MRP pumps, and isaccumulated in cells in the presence of ABC transporter inhibitor.This assay was carried out according to El-Readi et al. (7) usingverapamil (20 μM) as a standard P-gp and MRP1 inhibitor (9). Statistical analysis. GraphPad Prism™ (Version 4.00) software(GraphPad Software, Inc., San Diego, CA, USA) was used. All dataare expressed as the mean±standard error of the mean. Eachtreatment was tested in triplicate in each experiment, which in turnwas carried out about three times. One-way analysis of variance(one-way ANOVA), followed by Dunnett’s multiple comparisonpost test, was used to analyze the difference between treatments.The concentration–response curves were obtained by the Hillequation, according to the software instructions. ResultsCytotoxicity. Under our experimental conditions, HCAsignificantly reduced cell viability although at much higherconcentrations than those of the positive control doxorubicin(Table I).Combination assay. The combination of doxorubicin andHCA significantly reduced cell viability and increased thecytotoxicity of doxorubicin, as shown by the reversal ratioanalysis (Table I). In particular, for Caco-2 cells, the IC50value for doxorubicin was statistically reduced by about 3-and 7-fold in combination with HCA at IC10 (100 μM) andIC20 (181 μM) concentrations, respectively (Figure 2A,Table I). Likewise, for CCRF/CEM cells, the cytotoxicpotency of doxorubicin was significantly increased incombination with HCA: the IC50 value of doxorubicin wasreduced by about 5- and 50-fold in combination with IC10(50 μM) and IC20 (100 μM) concentrations of HCA (Figure2B and Table I). Viability of  the resistant CEM/ADR5000subline was also significantly affected by the combination(Figure 2C), with an increase of doxorubicin cytotoxicity ofapproximately 4- and 8-fold in combination with the IC10(100 μM) and IC20 (141 μM) concentrations of HCA (TableI). According to CI, the isobologram analysis highlighted asynergistic interaction between HCA and doxorubicin for allthe cell lines tested (Figure 3).Rhodamine 123 efflux assay. In Caco-2 cells, HCA increasedRho123 fluorescence in a statistically significant andconcentration-dependent manner with respect to the negativecontrol. At the same concentration of the positive controlverapamil (20 μM), about 35% increase in fluorescenceintensity was found. This highlights that a greater amount ofRho123 was retained in the cell, likely due to the inhibitionof the efflux pumps by HCA (Figure 4A). Conversely,Rho123 efflux was not affected by HCA in the CCRF/CEMcell line (Figure 4B). A fluorescence increase, similar to thatof verapamil, was observed in CEM/ADR5000 cells, thussuggesting possible P-gp pump inhibition (Figure 4C). DiscussionCinnamaldehyde has been found to possess beneficialproperties for human health, but its clinical application islimited due to its poor chemical stability and rapid clearance(6). Therefore, some alkyl-derivatives of cinnamaldehydehave been developed in order to make the molecule lessreactive and more stable. In the present study, thesemisynthetic derivative HCA was investigated for itspotential chemosensitizing properties in human cancer cells. The present results show that HCA itself has a lowcytotoxicity against the cancer cells tested: in contrast,cinnamaldehyde and its 2'-modified analogs were found topossess interesting antiproliferative activities (10). From astructural point of view, these molecules are characterized bythe presence of a propenal in their cinnamal skeleton that formsa Michael acceptor group (11). A number of compoundscontaining a Michael acceptor group have been shown toANTICANCER RESEARCH 36: 3347-3352 (2016)3348Figure 1. Chemical structure of α-hexylcinnamaldehyde.possess anticancer activity by interacting with cysteine residuesin different cellular targets: the reactivity of the Michaelacceptor moiety and lipophilicity have been proposed asstructural features for their anticancer properties (12). In thesemi-synthetic derivative HCA, the inclusion of an alkyl chainat α-β unsaturated double bond reduces the molecule'sreactivity due to the inductive effect of the alkyl group, whichstabilizes the negative charge in the intermediate carbanion.Accordingly, the presence of an α-hexyl chain in HCAstabilizes the lead compound structure (13), likely reducing theDi Giacomo et al: Chemosensitizing Properties of α-Hexylcinnamaldehyde3349Table I. Quantitation of synergistic, additive, and antagonistic interactions of the combination of α-hexylcinnamaldehyde (HCA) at inhibitoryconcentrations (IC)10 and IC20 (μM) with the anticancer drug doxorubicin (DOX) against Caco-2, CCRF/CEM and CEM/ADR5000 cells. For eachexperiment, the values for replicates were averaged and the values were expressed as the mean±SEM (n=9).Caco-2 CCRF/CEM CEM/ADR5000Drug IC50 (RR) CI r IC50 (RR) CI r IC50 (RR) CI r HCA 325.2±1.12 - 0.99 205.7±1.18 - 0.99 274.7±1.10 - 0.99DOX 5.3±0.50 - 0.99 0.4±0.01 - 0.99 87.7±1.2 - 0.99DOX + HCA IC10 1.9±0.16* (2.8±0.14) 0.7±0.01 0.97 0.08±0.02* (5.0±0.20) 0.44±0.004 0.95 20.3±0.13* (4.3±0.36) 0.6±0.01 0.97DOX + HCA IC20 0.8±0.06* (6.5±0.52) 0.7±0.02 0.94 0.008±0.0003* (50.9±1.58) 0.51±0.01 0.97 10.4±0.07* (8.4±0.30) 0.6±0.01 0.97RR: Reversal ratio, the ratio between the IC50 for DOX alone and DOX plus HCA; CI: combination index: 1, additive effect; <1, synergism; >1,antagonism; r: correlation coefficient for the regression line, calculated from the median-effect plots according to the basic mass-action principle(4).*Significantly different from the IC50 for DOX alone (p<0.01; ANOVA + Dunnett’s comparison multiple post test). Figure 2. Concentration–response curves for the cytotoxicity of doxorubicin (DOX) alone (black line) and in combination with α-hexylcinnamaldehyde (HCA) at inhibitory concentrations (IC)10 and IC20 against Caco-2 (A), CCRF/CEM (B) and CEM/ADR5000 (C) cells. Valuesare expressed as the mean±SEM (n=9). Figure 3. Isobologram analyses of the combination of doxorubicin (DOX) with with α-hexylcinnamaldehyde (HCA) at inhibitory concentrations(IC)10 (dark gray line) and IC20 (light gray line) against Caco-2 (A), CCRF/CEM (B) and CEM/ADR5000 (C) cells. The IC50 concentrations of DOXand HCA are plotted on the x and y axes, respectively. The line connecting these two points represents the additive effect; points located below andabove the line indicate a synergistic and antagonistic effect, respectively.Michael acceptor reactivity and its cytotoxicity. In contrast,these data suggest a possible role of HCA as a chemosensitizerwhen tested in combination with anticancer drugs, particularlydoxorubicin. This compound is a P-gp substrate, hence adifferent cytotoxicity profile in our cell lines could be expected(14). Lacking in ABC transporters, CCRF/CEM cells arehighly sensitive to doxorubicin alone because it is accumulatedin these cells and its cytotoxicity is induced at very lowconcentrations. Conversely, in Caco-2 and CEM/ADR500cells, the expression of ABC transporters (mainly P-gp) reducesthe potency of doxorubicin, hence higher concentrations arerequired. Under our experimental conditions, the presence ofHCA increased the potency of doxorubicin similarly in bothCEM/ADR5000 and Caco-2 lines, allowing us to hypothesizethat HCA acts by inhibiting the P-gp pump. However, resultsfrom Rho123 efflux assay suggest that HCA can interfere withABC transporter functionality, although not in a pump-specificmanner. Great synergism of HCA and doxorubicin was alsofound in CCRF/CEM cells (lacking in ABC transporters): thissuggests that HCA can indirectly inhibit efflux pumps. In thiscontext, it seems likely that non-specific mechanisms could beresponsible for the reduction of ABC transporter function. Insupport of this, compounds containing an α,β-unsaturatedmoiety have been reported to modulate MRP1- and MRP2-mediated transport via direct (chemical interaction) or indirect(formation of glutathione conjugates which can competitivelyinhibit MRP1 and MRP2 or glutathione depletion) mechanisms(5). Cinnamaldehyde is also known to indirectly inhibit MRP1-mediated transport by forming glutathione conjugates (5). Many MDR-reversing compounds have also been found toalter membrane fluidity and permeability, thereby modifyingmany aspects of transporters, including stability, conformation,and function: this represents a successful mechanism forreversing MDR by reducing the efflux of cytotoxic drugs. Forthis purpose, HCA has been found to deeply interact with abiomembrane model of dimyristoylphosphatidyl-cholinemultilamellar vesicles, to reduce phospholipid interaction,thereby altering the membrane properties (15). This alterationcan be potentiated in the presence of other lipophiliccompounds (such as doxorubicin), that can be trapped inbiomembranes and interfere with both phospholipids andmembrane proteins, resulting in a modulation of proteinfunction (16). Thereafter, the inhibition of the ABC transporterfunction by HCA could be due to its ability to block effluxpumps as a consequence of destabilization of biomembranestructure.In conclusion, the present results highlight that HCApossesses interesting chemosensitizing properties due tointerference with ABC transporter function, possibly as aconsequence of an alteration of membrane permeability, thussuggesting a possible involvement of non-classical MDRmechanisms in its chemosensitizing properties.AcknowledgementsThis work was supported by the Enrico and Enrica Sovena Foundation(Italy).References1 Kartal-Yandim M, Adan-Gokbulut A and Baran Y: Molecularmechanisms of drug resistance and its reversal in cancer. CritRev Biotechnol 24: 1-11, 2015.2 Chou TC: Theoretical basis, experimental design, andcomputerized simulation of synergism and antagonism in drugcombination studies. Pharmacol Rev 58: 621-681, 2006. Erratumin: Pharmacol Rev 59: 124, 2006.3 Saraswathy M and Gong S: Different strategies to overcomemultidrug resistance in cancer. Biotechnol Adv 31: 1397-1407,2013.4 Di Giacomo S, Mazzanti G, Sarpietro MG and Di Sotto A. α-Hexylcinnamaldehyde inhibits the genotoxicity of environmentalpollutants in the bacterial reverse mutation assay. J Nat Prod 77:2664-2670, 2014.ANTICANCER RESEARCH 36: 3347-3352 (2016)3350Figure 4. Effect of α-hexylcinnamaldehyde (HCA) on rhodamine 123 efflux in Caco-2 (A), CCRF/CEM (B) and CEM/ADR5000 (C) cells. Values areexpressed as the mean±SEM (n=9). VER, Verapamil. Statistically significantly different at *p<0.05, **p<0.01 and ***p<0.001 from VER, and at#p<0.001 from the negative control.5 Wortelboer HM, Usta M, van Zanden JJ, van Bladeren PJ,Rietjens IM and Cnubben NH: Inhibition of multidrug-resistanceproteins MRP1 and MRP2 by a series of α,β-unsaturatedcarbonyl compounds. 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J Med Chem 52:5311-5322, 2009.10 Gan FF, Chua YS, Scarmagnani S, Palaniappan P, Franks M,Poobalasingam T, Bradshaw TD, Westwell AD and Hagen T:Structure–activity analysis of 2'-modified cinnamaldehydeanalogues as potential anticancer agents. Biochem Biophys ResCommun 387: 741-747, 2009.11 Di Sotto A, Maffei F, Hrelia P, Di Giacomo S, Pagano E,Borrelli F and Mazzanti G: Genotoxicity assessment of somecosmetic and food additives. Regul Toxicol Pharmacol 68: 16-22, 2014.12 Muhammad Y and Kerr S: Comparative effects ofcinnamaldehyde and cinnamyl-containing compounds on theviability of two human melanoma cell lines, SK-MEL19 andSK-MEL23. S FASEB J 28: LB611, 2014.13 Roberts DW and Patlewicz G: Mechanism based structure-activity relationships for skin sensitisation–the carbonyl groupdomain. SAR QSAR Environ Res 13: 145-152, 2002.14 Kannan P, Brimacombe KR, Kreisl WC, Liow JS, Zoghbi SS,Telu S, Zhang Y, Pike VW, Halldin C, Gottesman MM, Innis RBand Hall MD: Lysosomal trapping of a radiolabeled substrate ofP-glycoprotein as a mechanism for signal amplification in PET.Proc Natl Acad Sci USA 108: 2593-2598, 2011.15 Sarpietro MG, Di Sotto A, Accolla ML and Castelli F:Interaction of α-Hexylcinnamaldehyde with a biomembranemodel: a possible MDR-reversal mechanism. J Nat Prod 78:1154-1159, 2015.16 Eid SY, El-Readi MZ and Wink M: Carotenoids reversemultidrug-resistance in cancer cells by interfering with ABCtransporters. Phytomedicine 19: 977-987, 2012.Received April 12, 2016Revised May 27, 2016Accepted May 31, 2016Di Giacomo et al: Chemosensitizing Properties of α-Hexylcinnamaldehyde3351

α-Hexylcinnamaldehyde synergistically increases doxorubicin cytotoxicity towards human cancer cell lines

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α-Hexylcinnamaldehyde synergistically increases doxorubicin cytotoxicity towards human cancer cell lines

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