[CANCER RESEARCH 49, 3917-3921. July 15, 1989|

Modifying Effect of Cinnamaldehyde and Cinnamaldehyde Derivatives on CellInactivation and Cellular Uptake of ciVDiamminedichloroplatinuni(II) inHuman NHIK 3025 Cells1

John M. Dornish,2 Erik O. Pettersen, and Reidar Oftebro

Department of Tissue Culture, Institute for Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo 3, Norway

ABSTRACT

The effect of Cinnamaldehyde and various Cinnamaldehyde derivativeson cell inactivation induced by cis-diamntinedichloroplatinum(II) (c/s-DDP) was investigated using human NHIK 3025 cells in culture. Cellinactivation was measured as a loss in the ability of single cells to giverise to macroscopic colonies following drug treatment. It was found thatCinnamaldehyde and a-chlorocinnamaldehyde potentiated the cell-inactivating effect when used simultaneously with m-DDP without increasingthe amount of cell-associated platinum. In contrast, a protective effectwith respect to cell inactivation was found when cells were treated withc/s-DDP in combination with hydrocinnamaldehyde or a-methylcinna-maldehyde. At higher concentrations (>1 HIM)all Cinnamaldehyde derivatives reduced cellular uptake of cw-DDP. Therefore, while protectionfrom cis-DDP-induced cell inactivation involves reduced platinum uptake,potentiation by Cinnamaldehyde and Cinnamaldehyde derivatives does notseem to be due to an increase in intracellular platinum. We propose thatCinnamaldehyde may compete with ciî-DDPin nucleophilic additionreactions involving intracellular sulfhydryls.

INTRODUCTION

Nontoxic doses of Cinnamaldehyde have previously been reported to potentiate the cell-inactivating effect of c/s-DDP' in

human NHIK 3025 cells (1). This effect was found to occuronly when Cinnamaldehyde and c/s-DDP were given simultaneously. It occurred, however, irrespective of the phase of thecell cycle within which treatment was started. The corresponding cinnamic acid and cinnamyl alcohol were found to have nopotentiating effect on cell survival following combination treatment of cells with c/s-DDP (1).

By comparison, previous reports (2, 3) have shown thatbenzaldehyde and the vitamin B6 aldehydes pyridoxal and pyr-idoxal 5'-phosphate protect cells from the inactivating effect of

c/s-DDP. It appears that benzaldehyde inhibits cellular uptakeof c/s-DDP by forming a Schiff base with cell membrane aminogroups (3, 4). From this one might speculate on the possibilitythat Cinnamaldehyde could act by increasing the amount of cell-associated platinum. This is, however, not the case since theuptake of platinum is unchanged in the presence of nontoxicdoses of Cinnamaldehyde (1).

By studying the structure-activity relationship of various cin-namaldehyde derivatives, we may approach an understandingof the mechanism for drug potentiation by these agents. In thispaper, we investigate the effect of various chemical substitutionsand their position on the Cinnamaldehyde molecule with respectto potentiation of c/s-DDP-induced cell inactivation.

Received 9/7/88; revised 2/24/89; accepted 4/13/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by grants from the Norwegian Cancer Society.2Fellow of the Norwegian Cancer Society. To whom requests for reprints

should be addressed.3The abbreviations used are: c/s-DDP, CK-diamminedichloroplatinum(II); SH,

sulfhydryl.

MATERIALS AND METHODS

Cell Line and Drug Treatment. Cells of the human line NHIK 3025,established from a cervical carcinoma in situ (5, 6), were cultivated asmonolayers in Medium E2a (7) supplemented with 20% human and10% horse serum. The cells were recultured every second or third dayto ensure continuous, exponential growth. Cells were trypsinized, anda known number was seeded into 60- x 15-mm style plastic Petri dishes.Dishes were kept in a CO2 incubator at 37°C.Drugs were added to the

cells as soon as these had attached to the bottom of the dishes, 2 h afterbeing seeded, by replacing the medium in the dishes with mediumcontaining the desired concentrations of drugs. Following the drugtreatment period, the dishes were rinsed with warm (37°C)Hanks'

balanced salt solution before fresh medium was added. After 12 to 14days, colonies of cells were fixed in ethanol and stained with méthylèneblue. Only colonies containing more than 40 cells were scored assurvivors, and data are expressed as the surviving fraction, i.e., thefraction of cells not inactivated by treatment and giving rise to macroscopic colonies relative to control cells.

Atomic Absorption Spectroscopy. Analysis of cell-associated platinum was performed using a Varian SpectrAA-30 atomic absorptionspectrometer fitted with a GTA-96 graphite tube atomizer. Cells weretreated in suspension with drugs, then washed in phosphate-bufferedNaCl solution, and finally taken up into concentrated UNO. as described previously (1). Aliquots representing 250,000 cells were placedin a graphite tube, and the atomic absorption signal measured at 265.9nm was registered. Platinum content was quantitated by running acalibration curve immediately before the samples.

Drugs. /rans-Cinnamaldehyde, hydrocinnamaldehyde, a-methylcin-namaldehyde, a-chlorocinnamaldehyde, 2-methoxycinnamaldehyde, 2-nitrocinnamaldehyde, 4-(dimethylamino)cinnamaldehyde, and acrolein(2-propenal) were purchased from Aldrich-Chemie GmbH & Co., Stein-heim. West Germany. The chemical structures are shown in Fig. 1. cis-Diamminedichloroplatinum (Platistin) was from Farmitalia CarloErba, Barcelona, Spain. Stock solutions of drugs were made up inHanks' balanced salt solution or directly in Medium E2a using ethanol

as a solubilizing agent fresh before each experiment. The concentrationof ethanol never exceeded 0.25%. After sterile filtration, drug dilutionsand drug combinations were made in Medium E2a immediately before

RESULTS

To test aldehyde-mediated modulation of c/s-DDP-inducedcell inactivation, we treated NHIK 3025 cells simultaneouslywith both c/s-DDP and Cinnamaldehyde derivatives. Fig. 2shows the results for two such derivatives displayed as thefraction of cells surviving a 2-h treatment with varying concentration of a-methylcinnamaldehyde alone up to 2.5 mM or incombination with 10 ¿IMc/s-DDP (A) and with a-chlorocinnamaldehyde alone or in combination with 5 MMc/s-DDP (B). a-Methylcinnamaldehyde alone had little effect on the survival ofexponentially growing cells, while 10 ^M c/s-DDP alone reduced the surviving fraction to 0.003. The simultaneous combination of c/s-DDP and a-methylcinnamaldehyde, however,displayed a protective effect; i.e., the surviving fraction of cellsis greater if a-methylcinnamaldehyde is present together withc/s-DDP than if c/s-DDP is present alone. In contrast, a-

3917

on May 23, 2018. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

cis-DDP PLUS C1NNAMALDEHVDE DERIVATIVES

s.

Fig. 1. The chemical structure of cinnamaldehyde (/), hydrocinnamaldehyde(2), (t-methylcinnamaldehydc (3). «-chlorocinnamaldehyde (4), «-methylcin-namaldehyde (5), 2-nitrocinnamaldehyde (6), 4-(dimethylamino)cinnamaldehyde(7), and acrolein (S).

0.1

0.01

0.001

DO-O O-

0123

CONCENTRATION 0F a-METHYLCINNAMALDEHYDE ( mM)

0.1

0.01

0.001

0.0001

0.00001

0.01 0.02 0.03 0.04 0.05

CONCENTRATION 0F a-CHLOROCINNAMALDEHYDE (mM)

Fig. 2. The surviving fraction of NHIK 3025 cells as a function of theconcentration of n-methylcinnamaldehyde (A) or n-chlorocinnamaldehyde (B)either alone (O) or in combination with c;'j-DDP (A), either IO pM for a-

methylcinnamaldehydc or 5 /»Mfor «-chlorocinnamaldehyde. Cells were treatedwith drugs or drug combinations for 2 h, then washed with Hanks' balanced salt

solution, and finally incubated 12 to 14 days in fresh medium for colony formation. Points, mean colony count from five replicate dishes per point; ears, SE. InB, the surviving fraction of cells treated with «-chlorocinnamaldehyde plus 5 /JMa'i-DDP normalized for the inactivating effect of «-chlorocinnamaldehyde alone

is shown (D).

chlorocinnamaldehyde potentiated the cell-inactivating effectof c/s-DDP. Although a-chlorocinnamaldehyde induces considerable cell inactivation alone, nevertheless, correcting the survival curve for c/s-DDP plus a-chlorocinnamaldehyde for thecell-inactivating effect induced by «-chlorocinnamaldehydealone indicates drug potentiation. Since a-chlorocinnamaldehyde induced toxic effects, a lower concentration of c/s-DDPwas used in drug combination experiments in order to keep thenumber of cells giving rise to colonies within acceptable limits.While the surviving fraction following 2-h treatment with 5 UM

cis-DDP alone was 0.16, the surviving fraction was reduced to0.013 (corrected) when 0.05 mivi a-chlorocinnamaldehyde wassimultaneously present.

Cell inactivation experiments of the type shown in Fig. 2were performed with all 1 cinnamaldehyde derivatives andacrolein. In order to be able to compare the effects induced bythese compounds directly, survival data were treated in thefollowing manner. The "relative surviving fraction," i.e., thesurviving fraction following treatment of cells with cis-DDPplus cinnamaldehyde derivative divided by the surviving fractionfollowing treatment with c/s-DDP alone, is plotted against thealdehyde concentration used. If an aldehyde induced a cell-inactivating effect alone, then the relative surviving fractionwas corrected for this. Data accumulated from experiments inwhich the cell-inactivating effect of cinnamaldehyde derivativeswas determined either alone or in simultaneous combinationwith c/i-DDP are compiled and redrawn together in Fig. 3. Thedashed line (indicating a relative surviving fraction of 1) represents a normalized cell survival following treatment with cis-DDP alone. If a cinnamaldehyde derivative potentiates the cell-inactivating effect of c/s-DDP, then the relative surviving fraction would be less than 1. Conversely, a cinnamaldehyde derivative which induces a protective effect would produce a relativesurviving fraction greater than 1 and, therefore, be above thedashed line.

From Fig. 3, two cinnamaldehyde derivatives, hydrocinnamaldehyde (Compound 2) and a-methylcinnamaldehyde(Compound 3), appear to induce a protective effect against cis-DDP-induced cell inactivation. The protective effect inducedby the aromatic aldehyde benzaldehyde is drawn in for comparison (data from Ref. 8).

One can also see from Fig. 3 that three compounds clearlypotentiate the cell-inactivating effect of cis-DDP. Cinnamaldehyde (Compound 1), which has previously been described (1),is used in this figure as a reference compound. Both a-chlorocinnamaldehyde (Compound 4) and the simple a,ß-unsaturatedcarbonyl compound acrolein (Compound 8) induce an evenstronger potentiating effect than cinnamaldehyde. On an aldehyde concentration basis, a-chlorocinnamaldehyde induces afar greater effect in combination with c/s-DDP than any of theother cinnamaldehyde derivatives tested.

100 -

ALDEHYDE CONCENTRATION (mM)

Fig. 3. The surviving fraction of cells treated with c/s-DDP plus cinnamaldehyde derivatives relative to the surviving fraction of cells treated with m-DDPalone as a function of the concentration of the aldehydes tested. Data wereobtained from survival curves of type shown in Fig. 2. Numbers refer to compounds as listed in Fig. \. B- •¿�-B, data for benzaldehyde from Ref. 8.

3918

on May 23, 2018. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

m-DDP PLUS CINNAMALDEHVDE DERIVATIVES

The remaining cinnamaldehyde derivatives, all possessingsubstituents on the phenyl ring, are seen to potentiate m-DDP-induced cell inactivation to a lesser degree than for cinnamaldehyde. Thus, placing substituents on the phenyl ring of cinnamaldehyde reduces the potentiating effect when these agentsare used in combination with m-DDP.

To test whether the potentiating effect on the cell-inactivatingpotential of m-DDP could result from a primary effect onplatinum uptake in human cells in culture, cell-associated platinum was determined in drug-treated cells by flameless atomicabsorption spectroscopy immediately following aldehyde plusCI5-DDP drug treatment. The results are shown in Table 1 asthe relative cell-associated platinum content derived by dividingthe platinum content of cells treated with 30 ^M m-DDP pluscinnamaldehyde derivative by the platinum content of cellstreated with m-DDP alone.

For cinnamaldehyde (Compound 1), it is apparent that theamount of cell-associated platinum decreases with increasingconcentrations of cinnamaldehyde in the drug combination.However, in the concentration range in which drug potentiationoccurs (from 0.1 to 0.5 mivi), equivalent amounts of platinumrelative to the platinum content of cells treated with m-DDPalone were found. Simultaneous treatment with m-DDP anda-chlorocinnamaldehyde (Compound 4) or acrolein (Compound 8) also displayed the same pattern of platinum uptake.

For cinnamaldehyde derivatives inducing a protective effect,hydrocinnamaldehyde and a-methylcinnamaldehyde (Compounds 2 and 3), Table 1 shows that the relative amount ofcell-associated platinum decreases with increasing aldehydeconcentrations. Similar results were also obtained followingtreatment of cells with 2-methoxy- and 2-nitrocinnamaldehyde(Compounds 5 and 6). Treatment of cells with m-DDP plus 4-(dimethylamino)cinnamaldehyde (Compound 7) caused littlechange in the amount of cell-associated platinum irrespectiveof aldehyde concentration.

In order to determine if the potentiation of m-DDP-inducedcell inactivation by cinnamaldehyde was due to a reactionbetween the two compounds, we preincubated solutions of cis-DDP with cinnamaldehyde at a 1:1 molar ratio. Cells werethereafter treated with concentrations of m-DDP in which thecinnamaldehyde concentration was diluted below the modifyingconcentration. In Fig. 4, the surviving fraction of NHIK 3025

cells is plotted against the concentration of m-DDP in cellculture medium. The data show that cell survival decreasesfollowing 2-h treatment with increasing concentrations of cis-DDP alone. Nearly identical survival was obtained when cis-DDP had been preincubated with cinnamaldehyde, indicatingthat potentiation of cw-DDP-induced cell inactivation by cinnamaldehyde is not due to extracellular adduci formation withplatinum but is cell mediated.

One may also ask the question if medium components are anecessary prerequisite for the expression of cinnamaldehydepotentiation. In Fig. 5, the survival of NHIK 3025 cells treatedwith Hanks' balanced salt solution for 2 h with m-DDP alone

or in combination with cinnamaldehyde is plotted against theconcentration of m-DDP. While the plating efficiency of NHIK3025 cells was not affected by 2-h incubation in Hanks' salt

solution, the cell-inactivating effect of m-DDP alone was reduced relative to incubation in serum-containing medium. Thismay reflect changes in the cell membrane or in m-DDP transport mechanisms when cells are incubated in a balanced saltsolution not containing amino acids or serum. However, eventhough drug exposure now occurred in Hanks' salt solution,

the simultaneous presence of 0.3 mivi cinnamaldehyde potentiated m-DDP-induced cell inactivation.

DISCUSSION

Based on our earlier finding that benzaldehyde protects cellsagainst m-DDP by reducing its uptake (2, 4), we first believedthat cinnamaldehyde was potentiating the cytotoxicity of m-DDP by increasing platinum uptake. This would also parallelthe effect described earlier for the pyrimidine analogue 1-propargyl-5-chloropyrimidin-2-one (9). However, measurements of cell-associated platinum content indicated that increased platinum uptake was not the cause for potentiation ofm-DDP-induced cell inactivation (Table 1; Ref. l). Even thevery strong potentiating effect of a-chlorocinnamaldehyde(Figs. 2 and 3) appeared without increasing platinum uptake(Table 1).

One type of reaction which may be responsible for the potentiation of m-DDP-induced cell inactivation, and is most distinctive for aldehydes possessing «,/3-unsaturated olefinic substituents, is adduct formation with cellular —¿�SHgroups, nota-

Table 1 Relative cell-associated platinum content of human NHIK 3025 cells treated with cis-DDP either alone or in simultaneous combination with variousconcentrations of cinnamaldehyde derivatives

NHIK 3025 cells in suspension were treated with 30 /IM c/s-DDP either alone or in simultaneous combination with various concentrations of cinnamaldehydederivatives for 2 h at 37°C.Immediately following drug treatment, the cells were centrifuged, washed with phosphate-buffered NaCI solution, and finally taken upinto HNOj. The platinum content of an aliquot representing 250,000 cells was determined by flameless atomic absorption spectroscopy as described in "Materialsand Methods." The numbers represent the relative cell-associated platinum content of cells, i.e., the platinum content following treatment with c/s-DDP pluscinnamaldehyde derivatives divided by the platinum content following treatment with c/s-DDP alone.

CompoundnameCinnamaldehydeHydrocinnamaldehyden-Methylcinnamaldehyde«-Chlorocinnamaldehyde2-Methoxycinnamaldehyde2-Nitrocinnamaldehvde4-(Dimethylamino)-cinnamaldehydeAcroleinCompoundno.123456Aldehyde

concentration(mM)0.0.0.0.0.0.0.08

1.0O.I0.960.970.921.090.920.960.910.05"0.060.050.050.030.060.050.95±0.030.251.010.970.941.060.930.950.970.050.050.010.030.020.040.040.95±0.030.50.88

±0.050.89±0.020.81±0.040.83±0.030.83±0.020.95±0.030.93±0.031.00

±0.0210.560.830.740.720.660.970.870.020.030.060.030.020.040.031.02±0.0420.52

±0.010.72±0.010.58±0.030.62±0.030.47±0.040.87±0.010.95±0.031.00

±0.0430.49

±0.010.54±0.020.51

±0.030.66±0.020.53±0.040.78±0.010.92±0.020.81

±0.04

Aldehyde concentration (mM)

0.005 0.025 0.05

«-ChlorocinnamaldehydeAcrolein

1.01.0

0.99 ±0.021.00 ±0.03

0.98 ±0.010.96 ±0.04

0.96 ±0.030.97 ±0.02

1.05 + 0.040.99 ±0.03

" Mean ±SE from at least two independent experiments.

3919

on May 23, 2018. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

ciî-DDPPLUS CINNAMALDEHYDE DERIVATIVES

0.1 -

0.01 -

0 8 10

CONCENTRATION 0F cis-DDP (MM)

Fig. 4. The surviving fraction of NHIK 3025 cells treated with cis-DDPpreincubated for 24 h with (A) or without (D) cinnamaldehyde as a function ofthe concentration of cis-DDP. The preincubation of cis-DDP was done in Hanks'

balanced salt solution at a concentration of 3.3 mivi.Cinnamaldehyde was addedat a 1:1 molar ratio, and solutions were incubated at 37"C protected from light.Dilutions were made in Medium E2a to the appropriate cis-DDP concentration.Cells were treated for 2 h, then washed in Hanks' salt solution, and finally

incubated in fresh medium 12 to 14 days for colony formation. Points, meancolony count from two independent experiments each having five replicate dishesper point; bars, SE.

0.001 r

O 10

CONCENTRATION OF cis-DDP (MM)

Fig. 5. The surviving fraction of NHIK 3025 cells treated in Hanks' balancedsalt solution with cis-DDP alone (D) or in combination with 0.3 mM cinnamaldehyde (A) as a function of the concentration of cis-DDP. Cells were treated for2 h with drugs alone or in combination in Hanks' salt solution (not completemedium), then washed, and reincubated in fresh serum-containing medium forcolony formation. Points, mean colony count from two independent experimentseach having five replicate dishes per point; bars, SE.

bly nonprotein sulfhydryls such as cysteine and glutathione (10,11). While reactions involving the aldehyde moiety probablyalso occur for these aldehydes, reactions involving additions tothe carbon-carbon double bond are of primary importance (10).

The aldehyde moiety is known to exert an electron-withdrawing inductive effect. This would activate the carbon-carbondouble bond toward nucleophilic addition by being able todisperse the negative charge which develops in such an additionreaction (10, 11). Nucleophilic addition to the /3-carbon couldtherefore occur with cellular sulfhydryls. One such example ofnucleophilic addition is the reaction of acrolein (which is structurally related to cinnamaldehyde) with sulfhydryls (11). (Areview of the biological effects of acrolein can be found in Ref.12.) Grafstrom et al. (13) and Krokan et al. (14) have demonstrated increased cytotoxic effects of acrolein relative to aliphatic aldehydes on human bronchial cells in culture. Moreimportant, their results link acrolein-induced cytotoxicity toreactions involving cellular sulfhydryl groups.

Addition of a phenyl moiety to position 3 of acrolein yieldscinnamaldehyde. Kurita et al. (15) have shown that cinnamaldehyde has the lowest energy in the lowest empty molecularorbital among a number of other conjugated aldehydes investi

gated. This indicates that cinnamaldehyde possesses very goodelectron acceptor properties. Moon and Pack (16) have suggested that cinnamaldehyde "traps" SH-containing amino

acids, and they indicate that such a reaction is more specific forcysteine than glutathione. Therefore, our assumption is thatcinnamaldehyde may potentiate the cell-inactivating effects ofcis-DDP due to addition reactions between cellular sulfhydryls

and cinnamaldehyde.The most probable mechanism is that cinnamaldehyde com

petes with cis-DDP for reaction with sulfhydryl groups, therebyincreasing the amount of cis-DDP available for reaction withthe cellular target, probably DNA. Faster reaction kinetics and/or greater formation constants for cinnamaldehyde plus —¿�SHthan for c/s-DDP plus —¿�SHwould effectively increase theintracellular concentration of c/s-DDP able to react with DNAby displacing c/s-DDP from SH adducts. Since the nonproteinsulfhydryl content of NHIK 3025 cells has been estimated tobe 6.4 mivi (17), it is doubtful that treatment for 2 h with 0.3mivi cinnamaldehyde could totally deplete intracellular sulfhydryls. Treatment with cinnamaldehyde, however, could lead todepletion of nonprotein sulfhydryls at specific intracellularsites, particularly in the vicinity of where DNA platinationoccurs (18). Although Hamilton et al. (19) and Andrews et al.(20) have shown that depletion of glutathione enhances c/s-DDP cytotoxicity, other reports find this not to be true (21,22). An increase in in vivo toxicity of c/s-DDP was found,however, in rats pretreated with diethyl maléate.Litterst et al.(23) conclude that a reduction of c/s-DDP binding —¿�SHsitesby diethyl maléatetreatment would allow more free c/s-DDPto interact with tissue binding sites.

The reactivity of the carbon-carbon double bond to nucleophilic addition is not only affected by the allylic aldehydepresent in cinnamaldehyde, but also by substituents on the a-carbon. An alkyl group, such as methyl in a-methylcinnamal-dehyde, releases electrons, thereby deactivating the conjugatedsystem towards nucleophilic addition (10, 11). If the deactiva-tion were strong enough, then only the aldehyde group wouldbe able to participate in cellular reactions in much the samemanner as for an arenic aldehyde, for instance, hydrocinnamal-dehyde. We find that, despite the presence of a conjugatedsystem, a-methylcinnamaldehyde induces a protective and nota potentiating effect when in combination with c/s-DDP (Figs.2 and 3). Hydrocinnamaldehyde also induces a protective effect,apparently via a reaction mechanism as for benzaldehyde, i.e.,Schiff base adduci formation (Fig. 3; Ref. 4). Furthermore, wefind that these cinnamaldehyde derivatives induce a protectiveeffect of the same order of magnitude as that induced bybenzaldehyde (2, 4).

Conversely, electron-withdrawing groups on the a-carbonwould activate towards nucleophilic addition (11). a-Chlorocin-namaldehyde induces a potentiating effect in combination withc/s-DDP at much lower concentrations than does cinnamaldehyde (Figs. 2 and 3). Activation of the carbon-carbon doublebond first by the aldehyde group and additionally by the chlorogroup could explain this result.

Finally, substituents on the phenyl ring of cinnamaldehydeapparently lead to slight deactivation of the carbon-carbondouble bond relative to cinnamaldehyde, irrespective of the typeof substituent on the phenyl ring (electron-withdrawing or-releasing) (Fig. 3). This deactivation results in either reducedreactivity towards addition of sulfhydryls or instability of theadduct formed. The end result appears as a reduction in thepotentiating effect of these aldehydes in combination with cis-DDP relative to cinnamaldehyde.

3920

on May 23, 2018. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

m-DDP PLUS CINNAMALDEHYDE DERIVATIVES

All aldehyde derivatives, with the exception of 4-(dimethyla-mino)cinnamaldehyde (Compound 7), when present at sufficient concentration (>1 HIM)reduced the amount of cell-associated platinum. We believe that high concentrations of cin-

namaldehyde derivatives act at the cell membrane in much thesame manner as benzaldehyde, that is, form Schiff base adductswith cell membrane amino groups leading to reduced platinumuptake. For cinnamaldehyde itself, the potentiation of m-DDPcytotoxicity was not due to extracellular reactions between thetwo compounds but required the presence of cells (Fig. 4). Inaddition, potentiation by cinnamaldehyde occurred irrespectiveof whether or not cells were incubated with m-DDP in completemedium or a minimal balanced salt solution (Fig. 5).

A clinical application of cinnamaldehyde to enhance thetherapeutic effect of m-DDP may be feasible, but would necessarily require adequate concentrations of cinnamaldehyde attumor sites. While the acute oral 50% lethal dose toxicity ofcinnamaldehyde in rats is 2220 mg/kg, and 1160 mg/kg inguinea pigs (24), chronic toxicity studies have shown that 2500ppm of cinnamaldehyde in the feed of animals are well toleratedand cause no long-term effects (25). Hepatic cell swelling andhyperkeratosis of the stomach epithelium were seen, however,at 10,000 ppm (25). Blood serum levels of cinnamaldehyde at0.3 HIM(equivalent to 39 mg/liter) may be tolerable over shorttimes, especially if induced simultaneously during m-DDPadministration.

ACKNOWLEDGMENTS

The skillful technical assistance of Charlotte Borka, Ursula PrehnHansen, and Athithan Kandiah is greatly appreciated. The NansenFund is acknowledged for providing financial support for the Perkin-Elmer Model 3600 data station used for cell survival analysis. TheNorwegian Research Council for Science and the Humanities (NAVF)provided funds for the VaríanSpectrAA-30 atomic absorption spectrometer.

REFERENCES

1. Dornish, J. M., Pettersen, E. O., and Oftcbro, R. Synergistic cell inactivationof human M UK 3025 cells by cinnamaldehyde in combination with cis-diamminedichloroplatinum(II). Cancer Res., 48: 938-942. 1988.

2. Dornish, J. M., Pettersen, E. O., Oftebro, R., and Melvik, J. E. Reductionof os-dichlorodiammineplatinum-induced cell inactivation by benzaldehyde.Eur. J. Cancer Clin. Oncol., 20: 1287-1293, 1984.

3. Dornish, J. M., and Pettersen, E. O. Protection from c/'i-dichlorodiam-

mineplatinum-induced cell inactivation by aldehydes involves cell membraneamino groups. Cancer Lett., 29: 235-243, 1985.

4. Dornish, J. M., Melvik, J. E., and Pettersen, E. O. Reduced cellular uptake

of c/i-dichlorodiammineplatinum by benzaldehyde. Anticancer Res., 6: 583-588, 1986.

5. Nordbye, K., and Oftebro, R. Establishment of four new cell strains fromhuman cervix. I. Exp. Cell Res., 58:458. 1969.

6. Oftebro, R., and Nordbye, K. Establishment of four new cell strains fromhuman cervix. II. Exp. Cell Res., 58: 459-460, 1969.

7. Puck, T. T., Cieciura, S. J., and Fisher, H. W. Clonal growth in vitro ofhuman cells with fibroblastic morphology. J. Exp. Med., 106:145-165.1957.

8. Dornish, J. M., Pettersen, E. O., and Oftebro, R. Modulation of m-dichlo-rodiammineplatinum (ru-DDP) induced cell inactivation by aldehydes: protection by benzaldehyde (Benz); potentiation by cinnamaldehyde (Cinn).Proc. Am. Assoc. Cancer Res., 29: 475, 1988.

9. Dornish, J. M., Pettersen, E. O., and Oftebro, R. Synergistic cell inactivationby c/5-dichlorodiammineplatinum in combination with l -propargyl-5-chlo-ropyrimidin-2-one. Br. J. Cancer, 56: 273-278, 1987.

10. Schauenstein, E., Esterbauer, H., and Zollner, H. Aldehydes in BiologicalSystems. Their Natural Occurrence and Biological Activities (Gore, P. H.,translator). London: Pion, Limited, 1977.

11. Esterbauer, H., Zollner, H., and Scholz, N. Reaction of glutathione withconjugated carbonyls. Z. Naturforsch., JOc: 466-473, 1975.

12. Izard, C.. and Liberman, C. Acrolein. Mutât.Res., 47: 115-138, 1978.13. Graftstrom, R. C., Dypbukt, J. M., Willey, J. C, Sundqvist, K., Edman, C,

Atzori, L., and Harris, C. C. Pathobiological effects of acrolein in culturedhuman bronchial epithelial cells. Cancer Res., 48: 1717-1721, 1988.

14. Krokan, H., Grafstrom, R. C., Sundqvist, K„Esterbauer, H., and Harris, C.C. Cytotoxicity, thiol depletion, and inhibition of O'-methylguanine-DNAmethyltransferase by various aldehydes in cultured human bronchial fibro-blasts. Carcinogenesis (Lond.). 6: 1755-1759, 1985.

15. Kurita, N., Miyaji, M., Kurane. R., Takahara, Y., and Ichimura, K. Antifun-gal activity and molecular orbital energies of aldehyde compounds from oilsof higher plants. Agrie. Biol. Chem., 43: 2365-2371, 1979.

16. Moon, K. H., and Pack, M. Y. Cytotoxicity of cinnamic aldehyde on leukemiaL12IO cells. Drug. Chem. Toxicol., 6: 521-535, 1983.

17. Nygaard, M. E. H., Dornish, J. M., Pettersen, E. O., and Oftebro, R.Sulfhydryl-related reduction of the mitotic inhibition and cell inactivationinduced by pyrimidine analogs. Anti-cancer Drug Design, 3: 15-24, 1988.

18. Smith, E., and Brock, A. P. An in vitro study comparing the cytotoxicity ofthree platinum complexes with regard to the effect of thiol depletion. Br. J.Cancer, 57: 548-552, 1988.

19. Hamilton, T. C, Winker, M. A., Louis, K. G., Batist, G., Behrens, B. C.,Tsuruo, T., Grotzinger, K. R., McKoy, W. M., Young, R. C., and Ozols, R.F. Augmentation of Adriamycin, melphalan. and cisplatin cytotoxicity indrug-resistant and -sensitive human ovarian carcinoma cell lines by buthio-nine sulfoximine mediated glutathione depletion. Biochem. Pharmacol., 34:2583-2586, 1985.

20. Andrews, P. A., Schiefer, M. A., Murphy, M. P., and Howell, S. B. Enhancedpotentiation of cisplatin cytotoxicity in human ovarian carcinoma cells byprolonged glutathione depletion. Chem.-Biol. Interact., 65: 51-58, 1988.

21. Richon, V. M., Schulte, N., and Eastman, A. Multiple mechanisms ofresistance to aVdichlorodiammineplatinum(II) in murine leukemia L1210cells. Cancer Res., 47: 2056-2061. 1987.

22. Hromas, R. A., Andrews, P. A., Murphy, M. P.. and Burns, C. P. Glutathionedepletion reverses cisplatin resistance in murine L1210 leukemia cells. CancerLett., 34: 9-13, 1987.

23. Litterst, C. L., Bertolero, F., and Uozumi, J. The role of glutathione andmetallothionein in the toxicity and subcellular binding of cisplatin. In: D. C.H. McBrien and T. F. Slater (eds.), Biochemical Mechanisms of PlatinumAntitumour Drugs, pp. 227-254, Oxford, UK: IRL Press, Ltd., 1986.

24. Jenner. P. M., Hagan, E. C, Taylor, J. M., Cook, E. L., and Fitzhugh, O.G. Food flavourings and compounds of related structure. I. Acute oraltoxicity. Fd. Cosmet. Toxicol., 2: 327-343, 1964.

25. Hagan. E. C, Hansen, W. H„Fitzhugh, O. G., Jenner, P. M., Jones, W. I.,Taylor, J. M., Long, E. L., Nelson, A.A., and Brouwer, J. B. Food flavouringsand compounds of related structure. II. Subacute and chronic toxicity. Fd.Cosmet. Toxicol., 5: 141-157, 1967.

3921

on May 23, 2018. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1989;49:3917-3921. Cancer Res   John M. Dornish, Erik O. Pettersen and Reidar Oftebro  -Diamminedichloroplatinum(II) in Human NHIK 3025 Cells

cisDerivatives on Cell Inactivation and Cellular Uptake of Modifying Effect of Cinnamaldehyde and Cinnamaldehyde

  Updated version

  http://cancerres.aacrjournals.org/content/49/14/3917

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/49/14/3917To request permission to re-use all or part of this article, use this link

on May 23, 2018. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from