ICANCER RESEARCH56. 551-555. February 1. 1996]

ABSTRACT

The effects of in vivo administration of a pharmacologically toxic doseof the lipophific cationic compound, MKT-077, were investigated in selected vital organs of the rat. MKT-077 (15 mg/kg body weight), admin

istered by bolus i.v. injection every day for 5 days, did not detectablyinfluence rat heart and kidney mitochondrial respiration. Although thesame dosage of MKT-077 significantly decreased respiratory rates in ratliver mitochondria relative to untreated controls, complete recovery wasevident within 3 days following drug withdrawal. Whereas the niltochondrial DNA of rat kidney and liver appeared to be unaffected by MKT-077treatment, levels of heart mtDNA were noticeably less than control levelsin the immediate interval following drug administration. However, thislatter effect was partially reversed as early as 10 days following treatmentand completely reversed within a 30-day posttreatment period. Theseresults strongly suggest that a pharmacologically toxic dose of MKT-077minimally affects the overall functional integrity of mitochondria in such

critical, although highly vulnerable, tissues as the heart, liver, and kidney.

INTRODUCTION

MKT-077 (formerly known as FJ776) is a novel rhodacyanine dyefound to be selectively taken up by the mitochondria of carcinomacells (1) and demonstrated both in vitro and in vivo to displayanticarcinoma activity. MKT-077 is currently undergoing investigation as a potential anticancer agent in Phase I/il clinical trials.

In contrast to the vast majority of clinically available anticarcinomacompounds for which nuclear DNA is the primary molecular target,the lipophilic cation MKT-077 pinpoints the mitochondria of tumorcells and subsequently influences the morphological and functionalintegrity of these structures (1—4).Delocalization of the positivecharge of lipophilic cations enables them to pass through the hydrophobic barriers of plasma membrane and mitochondrial membranelipid bilayers, and the high negative charge existing inside the mitochondria strongly attracts the positively charged drug (5). The attraction of lipophilic cations is greater for tumor cell mitochondria thanfor the mitochondria of normal cells, primarily because the mitochondrial membrane potential of carcinoma cells is higher than that ofnormal epithelial cells (2, 3, 6, 7).

Among the lipophilic cations that display a concentration-dependent mitochondrial toxicity are rhodamine 123, which inhibits mitochondrial ATP synthesis at the level of F0F1 ATPase (8), and dequalinium chloride, which inhibits NADH-ubiquinone reductase (9).MKT-077 has been demonstrated to inhibit the mitochondrial respiration of cancer cells, most likely via a nonspecific perturbation of themitochondrial membrane, and to have a mild-to-moderately degradative effect on the mtDNA,2 but not nuclear DNA, of a variety ofcancer cell types (10). Since mitochondria are the primary sites of

Received 6/7/95; accepted 11/17/95.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 in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

@ To whom requests for reprints should be addressed, at Dana-Farber Cancer Institute,Division of Cellular and Molecular Biology, Harvard Medical School, 44 Binney Street,Boston, MA 02115.

2 The abbreviations used are: mtDNA, mitochondrial DNA; DNP, 2,4-dinitrophenol;

G3PDH, glyceraldehyde-3-phosphodehydrogenase.

AlP-generating aerobic metabolism, selective mitochondrial toxicityin carcinoma cells that results from the enhanced uptake and retentionof lipophilic cations provides the basis for the selective anticarcinomaactivity displayed by these compounds (1—3,6, 7). However, sincehigh concentrations of lipophilic cations can be toxic to all mitochondna, it is conceivable that in vivo administration of certain lipophiliccations at high concentrations and/or for prolonged periods of timemight induce adverse effects in the mitochondria of normal cells aswell as of the targeted carcinoma cells.

The purpose of this study was to investigate the in vivo effects of atoxic dose of MKT-077 on several selected vital tissues. This wasaccomplished by examining both mitochondrial bioenergetic functionand the integrity of mtDNA in the heart, liver, and kidneys ofSprague-Dawley rats treated with a previously determined toxic doseof MKT-077. Our results, which indicate a partial yet completelyreversible impairment of mitochondrial function in several critical, yethighly vulnerable, rat organs, have important positive implicationswith regard to the clinical use of MKT-077 in the treatment ofcarcinoma.

MATERIALS AND METHODS

Drug Treatment for Analysis of Mitochondrial Respiratory Activityand Measurement of mtDNA Levels. FemaleSprague-Dawleyrats,averageweight of 120—125g, received iv. injections of a bolus dose of 15 mgMKT-0771kgbody weight dissolved in D5W solution every day for 5 days.Control rats received iv. injections of a bolus dose of the vehicle, D5Wsolution, every day during this 5-day period. Following sacrifice, the liver,heart, and kidneys were removed for analysis of mitochondrial respiratory

activity on the fifth day of treatment, after 3 days of recovery followingtreatment, and after 10 days of recovery following treatment. For analysis ofmtDNA levels, the heart and kidney were removed from animals receiving iv.injections on the fifth day of treatment, after 3 days of recovery followingtreatment, after 10 days of recovery following treatment, and after 30 days ofrecovery following treatment. The liver was removed on the fifth day oftreatment, after 2 days of recovery following treatment, and after 22 daysof recovery following treatment.

Isolation of Mitochondria. The liver, kidneys, and heart of MKT-077-treated and control rats were excised immediately following the sacrifice of theanimal and placed in the appropriate ice-cold isolation medium. Liver mitochondria were isolated by differential centrifugation at 4°Cas describedpreviously (11). Briefly, tissue was minced and homogenized in STE (250 mMsucrose, 1 mM Tris-HC1, and 1 mM EDTA, pH 7.4), 20% w/v, and centrifugedat 600 x g. The mitochondrial pellet was resuspended and washed twice inSm, followed by one wash in ST (250 mi@isucrose, 1 mMTris-HC1,pH 7.4),and resuspension of the final pellet in ST. Mitochondria were isolated fromwhole kidney tissue according to the same procedure, except that there wereonly two washes, one in STE and one in ST.

Mitochondria were isolated from heart ventricular tissue (bottom 2/3 of

organ) by a modification of the procedure by Palmer et al. (12). Heart tissue

was placed in ice-cold buffer I (10 mM KCI, 50 mM 4-morpholinepropanesul

fonic acid, and 2 mMEGTA, pH 7.4, plus 0.2% BSA) and rinsed 5—10timesto remove blood. The tissue was quickly weighed, minced finely with a razorblade, and placed in a beaker with ice-cold buffer I at a final concentration of10 ml buffer/kg tissue. Nagarse (5 mg/g wet weight tissue) was added to thebeaker, and the mixed solution was allowed to sit at 4°Cfor 2 mm. The tissue

was homogenized using a Polytron, and the resulting homogenate was dilutedtwo times with buffer I and centrifuged at 5000 x g for 5 mm. After discarding

551

In Vivo Administration of MKT-077 Causes Partial yet Reversible Impairment of

Mitochondrial Function

Ellen L. Weisberg, Keizo Koya, Josephine Modica-Napolitano, Yang Li, and Lan Bo Chen1

Dana-Farber Cancer Institute, Division of Cellular and Molecular Biology, Harvard Medical School, Boston, Massachusetts 02115 [E. L W., K. K., I'. L, L B. C.], and TuftsUniversity, Department of Biology, Medford, Massachusetts 02155 [J. M-N.]

Research. on February 12, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

DNA was partially depunnated in 0.25 NHC1for 10 mm, treated with 0.4N NaOH-0.6 M NaC1 for 30 mm for hydrolysis of the phosphodiester backbone,

and finally neutralized in 1.5 M NaCl-0.5 M Tris-HC1 (pH 7.5). DNA wastransferred to a GeneScreen Plus nylon membrane (NEN, Boston, MA) byovernight capillary blotting in lox SSC (0.15 Mtrisodium citrate and 1.5 MNaCl) according to the method of Southern (15).

Filters were soaked briefly in 2x SSC and prehybndized for 15—20mm at

42°Cin 50% deionized formamide (pH 7.5), 0.02 M Tris, 4x SSC, 1xDenhardt's solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02%BSA), 10%dextran sulfate, and 1%SDS. Filters were subsequently hybridizedin the same hybridization fluid containing 100 @g/mlsingle-stranded salmonsperm DNA and 1—2ng/ml of CT32P-labeled probe (3 x l0@ cpm4tg).

After labeling using the random 14-mer primer method (NEN, Boston,MA), probes were purified by elution over a Select-D, 0-50 spin column (5Prime—*3 Prime, Inc., Boulder, CO). Probes were boiled for 5 mm and placed

on ice for 2 mm prior to addition to hybridization fluid. Following overnighthybridization at 42°C, filters were autoradiographed using an intensifying

screen at —80°Cwith Kodak X-Omat AR film.6 Probes. The human mtDNA probe [excised and purified from the pGEM

72f(+) vector] consisted of a portion of the mitochondrial genome(8729—10254 bp) containing the coding region for the protein product COXIIIand for part of the protein products ATPase 6 and ND3. The 540-bp G3PDHprobe, a gift from Dr. Graham Barnard (University of Massachusetts MedicalCenter, Worcester, MA), was PCR amplified (16) from a cDNA clone usingthe primers 5'-ATGCICIQAAGGTGAAGCITCGG-3'and 5'-GGGTGCTAAGCAGTTGQT-3‘(17) and was used as an internal control for all experiments.

RESULTS

Effect of 15 mg/kg of MKT-077 on Average Body Weight ofRats. To effectively study the extent of mitochondrial impairmentthat might occur in normal tissues as a result of MKT-077 treatment,it was essential that we used the highest possible nonlethal dosage ofthe drug. This was determined to be 15 mg MKT-0771kg body weight,a maximally tolerable dose in the rat.3 We assessed the pharmacological effects of drug treatment in our study by measuring the total bodyweight of animals following treatment but just prior to being sacnficed. Fig. 1 demonstrates that treatment of animals with a dose of 15mg MKT-0771kg body weight each day for 5 days resulted in anaverage weight loss of 17%, whereas control rats gained an average of8% of their body weight over a span of 6 days. These results suggestthat both the drug dosage and treatment regimen used in this studywere sufficient to have significant adverse physiological impact.

Effect ofln Vivo Administration of MKT-077 on MitochondrialRespiratory Activity. We determined the effect of in vivo administration of MKT-077 on the respiratory activity of mitochondria isolated from the liver, kidneys, and heart of female Sprague-Dawley ratsthat had been treated with the compound. Fig. 2 shows that thesuccinate-induced, ADP-stimulated respiratory rate in mitochondriaisolated from the liver of rats treated with a bolus i.v. injection of 15mg MKT-0771kg body weight each day for 5 days is significantlylower than that of untreated controls. However, this inhibition isreversible since respiratory rates returned to and were maintained at(or above) control values in mitochondria isolated from the liver ofanimals sacrificed on days 3 and 10 following completion of the 5-daytreatment regimen. A similar effect was induced by MKT-077 onADP-stimulated respiration when a combination of glutamate andmalate was used as the respiratory substrate and on DNP-stimulatedrespiratory rates when either succinate or the combination of glutamate and malate was used as the respiratory substrate (data notshown).

Results show no significant effect of in vivo administration ofMKT-077 on respiratory activity in mitochondria isolated from the

3 K. Koya, unpublished data.

INFLUENCE OF MKT-077 ON MITOCHONDRIAL INTEGRITY

to0

0

0)>

to0)

.cC)0)

>.

0

Fig. 1. Effect of in vivo administration of MKT-077 on rat weight. Animals receivediv. injections for S days with 15 mg/kg body weight MKT-077. Simultaneously, controlanimals received iv. injections with vehicle over a span of 6 days. 0, weights recordedeach day for MKT-077-treated animals; 0, weights recorded each day for untreatedanimals.

the supematant, the pellet was resuspended in buffer I (to the previous volume)and centrifuged at 500 x g for 10 mm. The supernatant was collected andsaved, and the pellet was resuspended and centrifuged as in the previous step.The resulting supernatant was removed and combined with the previouslysaved supernatant, and the pellet was discarded. The combined supematantswere then centrifuged at 3000 X g for 10mm, and the mitochondrial pellet wasresuspended and washed two times in buffer I. The final pellet was resuspended in buffer II (buffer I minus BSA) to a final concentration of approximately 10 mg/ml. Protein was determined by the method of Lowry et al. (13).

Respiration. The respiratoryactivity of mitochondriaisolated from thevarious tissues of MKT-077-treated and control rats was measured polaro

graphically using a Clark oxygen electrode inserted into a l-ml water-jacketedchamber maintained at 30°C (14). The assay medium consisted of 225 mist

sucrose, 10 mrvipotassium phosphate (KH2PO4IK2HPO4), 5 mM MgC12, 10 mistKC1, 1 mM EDTA, and 10 mMTris-HCI (pH 7.4). An initial rate of oxygenconsumption (state 2) was recorded following the addition of a substrate, eitherglutamate plus malate (5 mMeach) or 10 mMsuccinate (+ 2 pg/mi rotenone),and a state 3 rate was recorded following the subsequent addition of 100 nmolof ADP. After a measurable state 4 rate (i.e., the rate after ADP is phosphorylated) was obtained, an 80 @LMconcentration of the uncoupling agent DNPwas added to obtain a rate of oxygen consumption in the absence of coupledoxidative phosphorylation.

Extraction of Total DNA from Tissue Samples. Rat heart, liver, andkidney tissue samples (0.25 g tissue) were ground in liquid nitrogen with aprechilled mortar and pestle and lysed overnight at 55°Cin 10 ml of 100 mMNaCl, 10 mM Tris-HC1 (pH 8.0), 50 mM EDTA (pH 8.0), 0.5% SDS, and 100

@g/mlproteinase K. Samples were extracted with an equal volume of phenol/

chloroform/isoamyl alcohol, spun for 10mm at 8000 rpm, extracted again withphenol/chloroform/isoamyl alcohol, and extracted once more with chloroform/

isoamyl alcohol. The aqueous phase was transferred to a fresh tube and treatedwith 200 @g/mlRNase at 37°C for 2 h. DNA was precipitated overnight at

—80°Cwith 2.5 volumes of prechilled 100% ethanol. DNA was spunat maximum speed in a microfuge for 30 mm, washed with 70% ethanol,air-dried, and resuspended in H20.

Southern Blot Hybridization. Genomic DNA extractedfrom rat heart,liver, and kidney tissues was rocked overnight at 4°Cto insure completedigestion. PvuII restriction enzyme (300 units; GIBCO) was used to digest 100

1.Lgof each sample. Digested DNA (10 @g)was size fractionated overnight at

20 V on a 0.21 xg/ml ethidium bromide-stained 0.8% agarose gel in Trisborate EDTA (pH 8.0). DNA was quantitated by OD2@ absorbance readings

on a spectrophotometer. Loadings between lanes were assessed by ethidiumbromide staining and hybridization of genomic DNA to a G3PDH probe.

0 1 2 3 4 5

Days of Treatment

552

Research. on February 12, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

INFLUENCE OF MKT-077 ON MITOCHONDRIALINTEGRITY

DISCUSSIONa'

I-a'

I. @.

@ as

.@ E

0

Fig. 2. Effect of in vivo administration of MKT-077 on ADP-stimulated respiratoryrates in rat liver mitochondria. 5 day treatment: mitochondria were isolated from the liverof animals that had received a bolus injection of 15 mg MKT-077/kg body weight eachday for 5 days (T), n = 5, and from untreated controls (C), n = 5. 5 day treatment, 3 dayrecover,': mitochondria were isolated from animals 3 days following completion of the5-day treatment regimen (T), n = 3, and from untreated controls (C), n = 3. 5 daytreatment, 10 day recovery: mitochondria were isolated from animals 10 days followingcompletion of the 5-day treatment regimen (T), n = 4 and from untreated control (C),n 4. ADP-stimulated respiration was calculated as the state 3 —state 2 rates (i.e., rateattributed solely to addition of ADP); bars. SE. Succinate was used as the respiratorysubstrate.

The MKT-077-associated impairment of tumor cell mitochondrialfunction and the marked influence of this drug on the morphologicalintegrity of tumor cell mitochondria have been well documented (10).Levels of drug required to inhibit both ADP- and DNP-stimulatedmitochondrial respiration in a dose-dependent manner were found tobe significantly less in a variety of cancer cell lines, including CX-l(colon cancer), MCF-7 (breast cancer), and CRL142O (pancreaticcancer), than in the normal green monkey kidney cell line, CV-!.Similarly, the fact that MKT-077 was demonstrated to have a selectivedegradative effect on the mtDNA of the aforementioned cancer celllines but no detectable effect on normal CV-! cells further suggests

T that this compound exhibits a discriminative toxicity toward cells of a5 day treatment10 day recovery malignant nature.

Selective mitochondrial toxicity resulting from increased mitochondrial uptake and retention of MKT-077 has been proposed to be thebasis for the selective antitumor effects of this compound. However,the potentially deleterious effects of high concentrations of MKT-077on the mitochondrial integrity of normal cells as well as of tumor cellscould, in theory, have a serious impact on the clinical use of thiscompound. Consequently, such a critical issue as this is in need ofimmediate and intensive investigation.

We chose to explore the possibility that, when administered in vivoat high doses and/or for prolonged periods of time, MKT-077 mightinduce mitochondrial toxicity in such normal tissues as the liver,heart, and kidneys of the rat. These organs were chosen in accord withprevious findings that show them to be the major sites of accumulation of MKT-077 following drug treatment. In addition, these vitaltissues traditionally exhibit the greatest risk for, and consequences of,drug toxicity.

The data obtained show that a dose of 15 mg MKT-0771kg bodyweight/day for 5 days had no effect on kidney or heart mitochondrialrespiration or DNA. In contrast, the same treatment caused a significant decrease in the respiratory rate of mitochondria isolated from ratliver (approximately 33% control values). However, complete recovery from this effect was evident at 3 days following completion of the

C T5 day treatment

C5 day treatment3 day recovery

kidney or heart (Fig. 3) of female Sprague-Dawley rats treated by i.v.injection with 15 mg MKT-0771kg/day for 5 days.

Effect of In Vivo Administration of MKT-077 on mtDNA Levels. We examined the effect of the in vivo administration of MKT-077on levels of mtDNA in the liver, heart, and kidneys of treated femaleSprague-Dawley rats. Total cellular DNA from tissues of animals thathad been treated by i.v. injection with 15 mg MKT-0771kg bodyweight and untreated controls was extracted on designated days anddigested with PvuII, which yields a single 16.5-kb band of mtDNA.Following an overnight run on an ethidium bromide-stained gel, theDNA was transferred to a nylon membrane and hybridized to eitherthe 32P-labeled mitochondrial probe or the G3PDH probe, which isrepresentative of nuclear DNA.

Fig. 4B, which shows the hybridization of rat mtDNA to the ratmtDNA probe, indicates no detectable effect by MKT-077 on mtDNAlevels in rat liver following 5 days of treatment. Further, levels ofmtDNA remained constant from 2 days to 22 days of recovery aftertreatment. Fig. 4A, which shows ethidium bromide-stained, PvuIIdigested total DNA, and Fig. 4C, which shows 32P-labeled G3PDH ineach DNA sample, together demonstrate equivalent loadings of DNAbetween each sample lane. In addition, these data indicate no detectable degradation of the nuclear DNA by MKT-077 in any of thetreatment groups versus the control groups. Similarly, there was noapparent effect by MKT-077 on mtDNA levels in rat kidney (data notshown).

In the rat heart, although there was no detectable effect by MKT077 on mtDNA levels immediately following 5 days treatment, therewas an apparent decrease relative to control by the third day ofrecovery (Fig. SB). Heart mtDNA levels in treated rats increasedslightly by the 10th day of recovery, and by the 30th day of recovery,treated and control levels of mtDNA were equivalent (Fig. SB). Fig. 5,A and D, which show ethidium bromide-stained, PvuII-digested totalDNA, and Fig. 5, C and F, which show 32P-labeled G3PDH in eachDNA sample, suggest equivalent loadings of DNA between the lanes.Since there is no detectable degradation of total cellular DNA and noapparent effect of MKT-077 on the nuclear gene, G3PDH, it can beassumed that the effect of MKT-077 on heart mtDNA is a selectiveeffect.

5 day treatment

I_I -@

c@o

rl)@

u@.@

Eo

Fig. 3. Effect of in vivo administration of MKT-077 on ADP-stimulated respiratoryrates in mitochondria isolated from rat kidney and rat heart. 5 day treatment: same asdescribed in Fig. 1. For kidney: (T), n = 7; (C), n = 7. For heart: (T), n = 2; (C), n 2.

553

Research. on February 12, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

INFLUENCE OF MKT-077 ON MITOCHONDRIAL INTEGRITY

A‘@ SQ

@ -@_

B (dA,@@AQ@@

16.5kb@@@

g-,@L__ C@i@

23.13 kb@

9.416 kb@

6.557kb @-

4.361 kb@

Fig. 4. Effect of in vivo administration of MKT-077 on mtDNA levels in rat liver. 6/8 L C, 6/8 L TR: total DNA extracted from liver tissue of animals that had received a bolusinjection of vehicle or 15 mg MKT-0771kg body weight, respectively, each day for 5 days. 6/10 L TR: total DNA extracted from liver tissue of animals 2 days following completionof 5-day treatment regimen. 6/30 L TR: total DNA extracted from liver tissue of animals 22 days following completion of 5-day treatment regimen. A, ethidium bromide-stainedPvuII-digested total tissue DNA (10 @sg/lane);B, hybridization of DNA shown in A with 32P-dCTP-labeled rat mtDNA probe; C, hybridization of DNA shown in A with32P-dCTP-labeled human G3PDH cDNA probe (internal control). Control, n = 1; treated, n = 1.

A@

‘@‘@@

23.13 kb@@@ ‘@-- ‘@?@@

9.416 kb@

6.5S7 kb@

4.361kb@

23.13kb@

9.416 kb @-

6.SS7kb@

4.361 kb@

Fig. 5. Effect of in vivo administration of MKT-077 on mitochondria DNA levels in rat heart. 5/6 H C, 5/6 H TR: control and treated samples, respectively, of total DNA extractedfrom heart tissue of animals that had received 5-day treatment regimen described in Fig. 4. 5/9 H C, 5/9 H TR: control and treated samples, respectively, of total DNA extracted fromheart tissue of animals 3 days following completion of 5-day treatment regimen. 5/16 H C, 5/16 H TR: control and treated samples, respectively, of total DNA extracted from hearttissue of animals 10 days following completion of 5-day treatment regimen. 6/6 H C, 6/6 H TR: control and treated samples, respectively, of total DNA extracted from heart tissueof animals 30 days following completion of 5-day treatment regimen. A and D, ethidium bromide-stained PvuH-digested total tissue DNA (10 @sgñane);B and E. hybridization of DNAshown in A and D, respectively, with 32P-dCTP-labeled rat mtDNA probe; C and F, hybridization of DNA shown in A and D, respectively, with 32P-dCTP-Iabeled human G3PDHcDNA probe (internal control). Control, n = 2; treated, n = 2.

554

()@()@

B@@

. .@.

@i@t::‘@@@@,•.“ @fSr, .@

c)le@ .@v A@@4

r

16@Skb-Ø@

VIVIYITI

c.)@ i@r ()@ i@r

@@ E

1)‘;;‘s;:;. @.@

-.- @.. @o

[Ii I@ 1&Skb@ @.,@ ...@ .@

@.. .@

i:'@1

Research. on February 12, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

INFLUENCE OF MKT-077 ON MITOCHONDRIAL INTEGRITY

treatment regimen and was stably maintained 10 days following drugremoval. Whereas MKT-077 treatment had no effect on liver andkidney mtDNA levels, it caused a slight decrease in the level ofmtDNA, but not nuclear DNA, in the heart as compared to theuntreated control. In contrast to the inhibitory effect on mitochondrialbioenergetic function, the effect of MKT-077 on heart mtDNAseemed to lag behind the treatment schedule and did not becomeapparent until 3 days following completion of drug treatment. By the10th day following drug removal, mtDNA levels were again approaching control values, and by the 30th day, there was no apparentdifference in mtDNA levels between treated versus control samples.The fact that mitochondrial toxicity by MKT-077 was detected only atthe level of mitochondnal respiration in the liver and only at the levelof mtDNA in the heart is interesting in that it suggests two separateand distinct sites of action of the drug.

The results of this study indicate that although an effectively highdose of MKT-077 administered in the rat did produce some adverseeffects in the mitochondria of the liver and heart, these effects werecompletely and stably reversible within a short period of time following completion of drug treatment. This finding has important positiveimplications with regard to the severity of potential side effects thatmay result from clinical use of the drug, since patients will undoubtedly receive well below the dose of MKT-077 comparable to that usedin these experiments. Further, these results are in stark contrast to theirreversible mitochondnal toxicity that was observed to result fromtreatment of patients with the nucleoside analogue and experimentalanti-hepatitis drug l-(2-deoxy-2-fluoro-1-@-D-arabinofuranosyl)5-iodouracil (FIAU) (18) and points to the very different mechanisms ofaction of these drugs.

REFERENCES

1. Koya, K., Li, Y., Wang, H., Ukai, T., Tatsuta, N., Kawakami, M., Shishido, T., andChen, L. B. MKT-077, a novel rhodacyanine dye in clinical trials, exhibits anticarcinoma activity in preclinical studies based on selective mitochondrial accumulation.Cancer Res., 56: 538—543, 1996.

2. Bernal, S. D., Lampidis, T. J., Summerhayes, I. C., and Chen, L. B. Rhodamine-123

selectively reduces clonal growth of carcinoma cells in vitro. Science (WashingtonDC),218: 1117—1119,1982.

3. Bernal, S. D., Lampidis, T. J., Mclsaac, R. M., and Chen, L. B. Anticarcinomaactivity in vivoof rhodamine 123,a mitochondrial-specific dye. Science (WashingtonDC), 222: 169—172,1983.

4. Weiss, M. J., Wong, J. R., Ha, C. S., Bleday, R., Salem, R. R., Steele, 0. D., andChen, L. B. Dequalinium, a topical antimicrobial agent, displays anticarcinomaactivity based on selective mitochondrial accumulation. Proc. Nail. Acad. Sci. USA,84: 5444—5448, 1987.

5. Johnson, L. V., Walsh, M. L., and Chen, L. B. Localization of mitochondria in livingcells with rhodamine 123. Proc. Natl. Acad. Sci. USA, 77: 990—994,1980.

6. Nadakavukaren, K. K., Nadakavukaren, J. J., and Chen, L. B. Increased rhodamine123 uptake by carcinoma cells. Cancer Res., 45: 6093—6099, 1985.

7. Modica-Napolitano, J. S., and Aprille, J. R. Basis for selective cytotoxicity ofrhodamine 123. Cancer Res., 47: 4361—4365, 1987.

8. Modica-Napolitano, J. S., Weiss, M. J., Chen, L. B., and Aprille, J. R. Rhodamine 123inhibits bioenergetic function in isolated rat liver mitochondria. Biochem. Biophys.Res. Commun., 118: 717—723,1984.

9. Anderson, W. M., Patheja, H. S., Delinck, D. L., Baldwin, W. W., Smiley, S. T., andChen, L. B. Inhibition of bovine heart mitochondrial and paracoccus denitrificansNADH-> ubiquinone reductase by dequalinium chloride and three structurallyrelated quinolinium compounds. Biochem. mt., 19: 673—685, 1989.

10. Modica-Napolitano, J. S., Koya, K., Weisberg, E., Brunelli, B. T., Li, Y., and Chen,L. B. Selective damageto carcinomamitochondriaby the rhodacyanineMKT-077.Cancer Res., 56: 544—550, 1996.

11. Aprille, J. R., and Austin, J. Regulation of the mitochondrial adenine nucleotide poolsize. Arch. Biochem. Biophys., 212: 689—699, 1981.

12. Palmer, J. W., Tandeler, B., and Hoppel, C. L. Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J. Biol.Chem., 23: 8731—8739, 1977.

13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurementwith the folin phenol reagent. J. Biol. Chem., 193: 265—275,1951.

14. Aprille, J. R., and Asimakis, G. K. Postnatal development of rat liver mitochondria:state 3 respiration, adenine nucleotide translocase activity, and the net accumulationof adenine nucleotides. Arch. Biochem. Biophys., 201: 564—575,1980.

15. Southern, E. M. Detection of specific sequences among DNA fragments separated bygel electrophoresis. J. Mol. Biol., 98: 503—517,1975.

16. Saiki, R. K., Scharf, S., Faloona, F. A., Mullis, K. B., Horn, G. T., Erlich, H. A., andAmhelm, N. Enzymatic amplification of @3-globingenomic sequences and restrictionsite analysis for the diagnosis of sickle cell anemia. Science (Washington DC), 230:1350—1354,1985.

17. Barnard, G. F., Staniunas, R. J., Mori, M., Puder, M., Jessup, M. J., Steele, G. D., Jr.,and Chen, L. B. Gastric and hepatocellular carcinomas do not overexpress the sameribosomal protein messenger RNAs as colonic carcinoma. Cancer Res., 53:4048—4052,1993.

18. Parker, W. B., and Cheng, Y. C. Mitochondrial toxicity of antiviral nucleosideanalogs. J. NIH Res., 6: 57—61,1994.

555

Research. on February 12, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

1996;56:551-555. Cancer Res   Ellen L. Weisberg, Keizo Koya, Josephine Modica-Napolitano, et al.   Impairment of Mitochondrial Function

Administration of MKT-077 Causes Partial yet ReversibleIn Vivo

  Updated version

  http://cancerres.aacrjournals.org/content/56/3/551

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/56/3/551To request permission to re-use all or part of this article, use this link

Research. on February 12, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from