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[CANCER RESEARCH 54, 4698-4702, September 1, 1994]

Effect of l,19-Bis(ethylamino)-5,10,15-triazanonadecane onHuman Tumor Xenografts1

M. Eileen Dolan,2 Matthew J. Fleig, Burt G. Feuerstein, Hirak S. Basu, Gordon D. Luk, Robert A. Casero, Jr.,

and Laurence J. Marton

Division of Hematologv/Oncologv. The University of Chicago Medical Center, Chicago, Illinois 60637 [M. E. D., M. J. FJ; Brain Tumor Research Center of the Department ofNeurological Surgery IH. S. B., B. G. F.Â¡,Department of Laboratory Medicine Â¡B.G. F.], and Department of Pediatrics [B. C. F.j, School of Medicine, University of California,San Francisco, California 94143: Gastroenterologe Research, Department of Veterans Affairs Medical Center, and The University of Texas Southwestern Medical Center, Dallas,Texas 75216 Â¡G.D. L.Â¡;Johns Hopkins Oncology Center Laboratories, Johns Hopkins School of Medicine, Baltimore, Maryland 21231 Â¡R.A. C.J: and Departments of Pathologyand Laboratory Medicine. Oncology, and Human Oncology, University of Wisconsin-Madison Medical School, Madison, Wisconsin 53706 [L. J. M.]

ABSTRACT

The polyamine analogue l,19-bis(ethylamino)-5,10,15-triazanonade-cane (BE-4-4-4-4), 5 mg/kg i.p.. was given twice daily on days 0-3 and7-10 (cycle 1) to nude mice with human malignant gliomas (SF-767 andU-87 Mdi. lung adenocarcinoma (A549), and colon carcinomas (HCT116and HT29). A second cycle of drug was given to mice with SF-767 andA549 tumors on days 42â€”45and 49-52. The maximum animal weight lossvaried between 4 and 12%, which was observed 10-15 days following the

initiation of treatment, but no overt toxic reactions were noted. TheSF-767 brain tumors were extremely responsive to BE-4-4-4-4 alone (3 of8 complete regressions after 2 cycles); however, the growth of the U-87MG brain tumor was only slightly inhibited by BE-4-4-4-4 treatment.

There was significant inhibition of tumor growth after treatment with onecycle of BE-4-4-4-4 in animals carrying the A549, HCT116, and HT29

tumors. At day 73, the growth of the A549 tumor was inhibited by 78 and89% following one or two cycles of BE-4-4-4-4, respectively. The mitotic

index of A549 tumors was 18 times greater in control mice than in thosetreated with BE-4-4-4-4 for one or two cycles 99 days after initiation oftreatment. 13-Bis(2-chloroethyl)-l-nitrosourea (BCNU) was given to micecarrying the U-87 MG or A549 tumors on day 4 (cycle 1) and day 46 (cycle

2) in the maximal tolerated dose of 50 mg/kg for BCNU alone and 40mg/kg for BCNU plus BE-4-4-4-4. BCNU alone significantly inhibited thegrowth of U-87 MG tumors but not the growth of A549 tumors. Treatmentwith the combination of BCNU and BE-4-4-4-4 was significantly betterthan BCNU alone for A549 tumors and better than BE-4-4-4-4 alone for

U87 tumors. However, in both animal groups treated with the combination, there was a significant weight loss, which was not observed foranimals treated with either agent alone. These data suggest a role forBE-4-4-4-4 in the treatment of brain, lung, and colon tumors.

INTRODUCTION

The polyamines spermidine and spermine and the diamine putres-

cine are biological cations that are present in all mammalian cells andare essential for normal growth processes (1). The activities of thepolyamine biosynthetic enzymes ODC3 and 5-adenosylmethionine

decarboxylase are regulated during the cell cycle and are induced byvarious trophic influences (1). The inhibition of polyamine biosynthesis is a potential target for chemotherapeutic intervention. DFMO,which inhibits ODC, has been studied extensively as an antineoplasticagent and as a chemopreventive agent (2, 3) and has shown antitumor

Received 2/14/94; accepted 7/6/94.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.

1This work was supported in part by Grants CA 47228 (to M. E. D.), CA 49409 (to

H. S. B.), CA 13525 (to H. S. B., B. G. F., and L. J. M.), CA 15206 (to G. D. L.), and CA58184, and CA 51085 (to R. A. C.) from the National Cancer Institute, the PattersonChair, and the National Brain Tumor Foundation.

2 To whom requests for reprints should be addressed, at Division of Hematology/

Oncology, 5841 S. Maryland Ave., Box MC2115, The University of Chicago MedicalCenter, Chicago, IL 60637.

3 The abbreviations used are: ODC, ornithine decarboxylase; DFMO. difluoromethyl-ornithine; BE-4-4-4-4, l,19-bis(ethylamino)-5,10,15-triazanonadecane; SSAT, spermi-dine/spermine-N'-acetyltransferase; BCNU, l,3-bis(2-chloroethyl)-l-nitrosourea; FBS,

fetal bovine serum.

activity in tissue culture, animal studies, and in clinical trials inmalignant glioma patients (4, 5). Although there have been someencouraging clinical results with DFMO, it has not shown muchpromise as curative therapy other than in some glial tumors (6). Someof the problems which may be responsible for the clinical ineffectiveness of DFMO include uptake and cellular compensatory mechanisms (7).

Recently, polyamine inhibitors have been designed to overcome theproblems associated with DFMO. Modified analogues of spermidineand spermine that deplete cellular polyamine pools and inhibit cellgrowth have been identified (8-12). Some of the effective agents areA^-bistethyOspermidine, A^X^bisiethyOspermine, W'X'-bis-

(ethyl)norspermine, and A^.A^-bisiethy^homospermine (12-17). Invivo studies using MALME-3M melanoma xenografts have indicatedthat A/1,A'11-bis(ethyl)norspermine produced tumor regressions, sup

pressed tumor regrowth during treatment, and sustained growth inhibition after treatment (12).

The mechanism by which polyamine analogues inhibit tumorgrowth is unclear. In some cell lines, the growth inhibitory effects ofpolyamine analogues are correlated with their ability to enhanceSSAT activity and to decrease cellular polyamines (17). Polyamineanalogues may also compete for intracellular binding sites of polyamines and inhibit their normal functions (11). Negatively chargednucleic acids are probable intracellular binding sites for polyamines.Evidence suggests that spermine bends specific DNA sequences before DNA condensation in vitro (18, 19). This bending and condensation of DNA may affect chromatin condensation in vivo. Polyamineaddition increases the extent of condensation of both naked DNA andDNA in chromatin and in nuclei isolated in vitro (20-23); polyamine

depletion also alters the chromatin structure in nuclei of cultured cells(24, 25).

Polyamine analogues that enter cells and compete with naturalpolyamines at intracellular binding sites that regulate cell growth mayact as antiproliferative agents (24, 26, 27). A new pentamine, BE-4-4-4-4, has a much greater affinity for DNA and is slightly less

effective at condensing and aggregating DNA than spermine (28).Thus, it could compete with spermine at growth regulatory sites onDNA but not allow functional bending or condensation of nucleicacid. Exposure of brain tumor cells to BE-4-4-4-4 depletes putrescine,

spermidine, and spermine, inhibits cell growth (28), and delaysgrowth of U-251 MG human glioma xenografts in nude mice.4 In

addition, both DFMO and BE-4-4-4-4 enhance the toxicity of BCNUin tissue culture (29),5 and BCNU alone or in combination with

DFMO has shown promising effectiveness in treating gliomas (4, 5,30). In this study, we tested the antitumor effects of BE-4-4-4-4 alone

4 C. J. Bergeron, L. J. Marton, K. Lamborn, H. S. Basu, A. Shirahata, K. Samejima,

and B. G. Feuerstein, Antitumor activity of l,19-bis-(ethylamine)-5,10,15-triazanonade-cane (BE-4-4-4-4) on human brain tumor xenografts, submitted for publication.

5 A. Sarkar, M. Pellarin, H. S. Basu, B. G. Feuerstein, L. J. Marton, and D. F. Deen,

unpublished observations.

4698

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POLYAMINE ANALOGUES IN HUMAN TUMORS

and in combination with BCNU against human glioma and lung andcolon tumor xenografts.

MATERIALS AND METHODS

Materials. BE-4-4-4-4 was synthesized by Drs. A. Shirahata and K. Same-

jirna at Josai University (Keyakidai, Sakado, Saitama, Japan; Ref. 28). BCNU(NSC 409962) was kindly provided by the Drug Synthesis and ChemistryBranch, Division of Cancer Treatment, National Cancer Institute (Bethesda,MD). SF-767 cells were obtained from the Brain Tumor Research Center at the

University of California, San Francisco. HT29 cells were a gift from Dr. L.Erickson, Loyola University Medical Center (Maywood, IL); U-251 MG cellswere obtained from the National Cancer Institute (Frederick, MD); and U-87

MG, A549, and HCT116 cells were obtained from American Type CultureCollection (Rockville, MD).

Animals. BALB/c (mt/nu) mice obtained from HarÃanSprague-Dawley

(Frederick, MD) were used in studies of HCT116 tumor xenografts. FemaleNIH Swiss random bred athymic mice obtained from the Frederick CancerResearch and Development Center (Frederick, MD) were used for all othertumor xenografts. All mice were maintained under a controlled 12-h light/12-h

dark cycle and allowed free access to water and food. Athymic mice werehoused in isolated cubicles in a high-efficiency particulate air-filtered envi

ronment. All supplies were sterilized before passage into the facility. Allanimal procedures adhered to the principles enumerated in the Guide for theCare and Use of Laboratory Animals.h

Cell Growth. Cells were grown in an environment of 5% CO2 in humidified air and were passaged weekly. SF-767 and U-87 MG cells were grown inDulbecco's modified Eagle's medium with 10% FBS, penicillin (100 units/

ml), streptomycin (100 fig/ml), nonessential amino acids (100 /AM),sodiumpyruvate (1 mM), and 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acidbuffer (10 HIM). A549 cells were grown in Ham's F-12K (Kaigns) supple

mented with 10% FBS, penicillin (100 units/ml), streptomycin (100 fig/ml),and L-glutamine (1 mM). HCT116 cells were grown in RPMI 1640 with 10%

FBS, penicillin (100 units/ml), and streptomycin (100 fig/ml). HT29 cells weregrown in minimal essential medium with 10% bovine calf serum,4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer (20 mM), sodiumpyruvate (1 mM), nonessential amino acids (200 fiM), L-glutamine (1 mM),

gentamicin (0.05 mg/ml), and biotin/B12.Tumor Growth. The mice received injections of tumor cells from one of

five human tumor cell lines: glioblastoma (SF-767, 2.5 X 10" or U-87 MG,7.9 X IO6); lung adenocarcinoma (A549, 2.7 X IO6); or colon carcinoma(HCT116, 10 X 10" or HT29, 10.2 x IO6). Body weight and tumor size were

monitored two times/week until the tumor reached a volume greater than 2000mm', the criterion for euthanasia.'' Two perpendicular tumor diameters, width

(the smallest dimension) and length (the largest dimension) were measuredwith calipers. Tumor growth data are expressed as tumor volumes (mm3)calculated from the following equation: length X width2 X 0.52 (31). Statis

tical analysis used was the Number Cruncher Statistical System developed byDr. Jerry Hitze (Kaysville, UT). All groups were compared using a two-sided

/ test.Drug Treatment. Treatment began 8, 14, 16, 17, and 32 days after mice

received injections of HT29, SF-767, U-87 MG, HCT116, and A549 cells,when tumors reached averages of 127, 67, 53, 11, and 74 mm3, respectively.

The effect of BE-4-4-4-4 was evaluated in all 5 tumor xenografts. BE-4-4-4-4

in 0.5 mg/ml saline vehicle (0.9% NaCl, pH adjusted to 7.4 with 100 mMNaHCOj) was given i.p. in a dose of 5 mg/kg (0.2 ml/20 g mouse) twice dailyon days 0, 1,2, 3, 7, 8, 9, and 10 (cycle 1). On day 42, SF-767 and A549tumor-bearing mice were treated with the same dose twice/day on days 42, 43,44, 45, 49, 50, 51, and 52 (cycle 2). Mice with U-87 MG, HT29, or HCT116tumors received only 1 cycle of BE-4-4-4-4. The dose schedule was based onprevious studies using tumor-bearing nude mice.4 BCNU solution was pre

pared in 10% ethanol/saline immediately before injection and was given i.p. ina dose of 50 mg/kg (5 mg/ml solution) for mice receiving BCNU alone and 40mg/kg (4 mg/ml) for mice receiving BE-4-4-4-4 with BCNU on day 4 (cycle

1) and day 46 (cycle 2). These were the maximum tolerated doses of BCNU

as determined by animal weight loss (32). Mice with SF-767 and HCT116tumors were randomized to receive vehicle alone (same schedule as BE-4-4-4-4) or BE-4-4-4-4 alone. Mice with A549, HT29, or U-87 MG tumors wererandomized to receive vehicle alone, BE-4-4-4-4 alone, BCNU alone, orBE-4-4-4-4 and BCNU in combination. Complete tumor regression was de

fined as no visible tumor. Animal toxicity was assessed by weight loss. Therewere no animal deaths in any group.

Histopathological Evaluation. On day 99, mice with A549 tumors weresacrificed, and tumors were cut into 2-11 pieces. Twelve to 18 sections of each

were made for histopathological examination.

RESULTS

SF-767 and U-87 MG Gliomas. There was a significant inhibitionof SF767 tumor growth observed for animals treated with BE-4-4-4-4

compared to control animals after 1 or 2 cycles (Table 1; Fig. LA).BE-4-4-4-4 treatment resulted in 3 of 8 complete regressions (cures)

after 2 cycles. The greatest weight loss (12%) was observed on day10; however, mice recovered and did not display any overt toxicreactions (Fig. IB).

The U-87 MG tumor xenografts were not as responsive as theSF-767 tumors to BE-4-4-4-4 alone (Fig. 2A). Although the growth

rate was slightly less in treated mice than in controls, this differencewas not significant (Table 1; Fig. Z4 ). BCNU had a dramatic effect ontumor growth (79% inhibition compared to controls); however, thecombination of BE-4-4-4-4 and BCNU (92% inhibition compared to

controls) was not significantly different than BCNU alone (Table 1;Fig. 2B). Weight loss was significantly greater for the combination ofBE-4-4-4-4 and BCNU than for either treatment alone. (Table 1;

Fig. 2, C and D).A549 Lung Carcinoma. BE-4-4-4-4 was effective against A549

tumor xenografts. It significantly inhibited tumor growth for up to 98days after initiation of treatment following either 1 or 2 cycles (Fig.3/4). The average tumor size at day 73 was larger in mice that receivedonly 1 cycle of BE-4-4-4-4 than those receiving 2 cycles (280 mm3versus 137 mm3), and both groups were significantly smaller thananimals receiving saline (1243 mm3) (Table 1). The average weight

Table 1 Effecr of BE-4-4-4-4 on the mean tumor volume of human iunior xenografis

Animal weight' Tumor volume'Tumor Treatment" Day n (g) P1 (mm' ) P1

SF767SalineBE-4-4-4-4U87MGSalineBE-4-4-4-4BCNUBE-4-4-4-4/BCNUA549SalineBE-4-4-4-4BE-4-4-4-4*BCNUBE-4-4-4-4/BCNUBE-4-4-4-4/BCNU*HCT116SalineBE-4-4-4-4HT29SalineBE-4-4-4-44242252525257373737373735656272788910108643743448824.7Â±0.623.2Â±0.823.2

Â±0.921.6Â±0.822.5Â±0.718.5Â±0.725.3

Â±0.823.6Â±0.924.2Â±1.925.4Â±0.423.0Â±0.120.7Â±1.125.7

Â±0.223.6Â±0.124.1

Â±0.422.5Â±1.30.160.190.520.00130.190.530.910.02911.01Â»0.00020.271405

Â±155508Â±1091999

Â±3881169Â±274414Â±310155

Â±921243

Â±307280Â±95137Â±161011Â±241182Â±4361Â±151153

Â±45247Â±91603

Â±2546.96Â±80().(XK)30.0930.00500.00120.0240.0160.560.0190.0340.00030.0078

h Committee on Care and Use of Laboratory Animals of the Institute of Laboratory

Resources, National Resource Council (DHEW publication No. 85-23, 1985).

" Animals were treated i.p. with saline or 5 mg/kg BE-4-4-4-4 twice daily for 4 days

on/3 days off/4 days on, starting on day 0 for cycle I and day 42 for cycle 2. All data isfor one cycle of drug except that denoted with an (*), which refers to 2 cycles of treatment,

n, number of animals in each group.' Data represent the mean animal weight (Â±SE) and mean tumor volume (Â±SE).'' Two-sided / test; compared to animals treated with saline.




20 40DAY

60

Fig. 1. Tumor growth rate of SF-767 xenografts treated with BE-4-4-4-4. Nude micecarrying SF-767 xenograft tumors were given i.p. injections of saline (â€¢)or 2 cycles of5 mg/kg b.i.d. of BE-4-4-4-4 (*) on day 0-3 and 7-10 (cycle 1) and days 42-45 and49-52 (cycle 2). A, tumor volume (mm3) for each group. B, animal weights for eachgroup. The doubling time of the tumor from 500 mm3 to 1(XK)mm3 was 11 days. Points,

the mean tumor volume and body weight for 8 mice per group; bars, SE.

loss (12%) was observed at 15 days for mice treated with 1 cycle, anda second weight loss (7%) was observed at 57 days for those treatedwith 2 cycles (Fig. 3C). The maximum tolerated dose of BCNUproduced little response in A549 tumors (Fig. 3B). The combinationof BE-4-4-4-4 with 40 mg/kg BCNU produced a markedly betterresponse than BCNU alone but was only slightly better than BE-4-4-4-4 alone. More weight loss (20%) occurred after 1 or 2 cycles of the

combined treatment compared to either treatment alone (Table 1; Fig.3D). Histopathological examination of A549 tumor xenograftsshowed an 18-fold decrease in the mitotic index of BE-4-4-4-4-treated

tumors compared to controls at day 99.HCT116 and HT29 Colon Carcinomas. Tumor growth inhibition

was evident at 10-14 days after initial treatment with BE-4-4-4-4 and

was maintained until day 56 in HCT116 tumor xenografts (Fig. 4/4).At day 56, animal weight loss was 8%, and tumor growth inhibitionwas 79% (Table 1; Fig. 4).

Significant growth inhibition was observed in HT29 tumors inresponse to BE-4-4-4-4 (Fig. 5A). On day 27, the growth inhibition

was 57% (Table 1). Again, all mice except controls had a transientweight loss (Fig. 5ÃŸ).

DISCUSSION

This report indicates that BE-4-4-4-4 has antitumor effects inhuman glioma, lung, and colon tumor xenografts. There was a sig-

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Fig. 2. Tumor growth rate of U-87 MG xenografts treated with BE-4-4-4-4 Â±BCNU.A and C. nude mice carrying U-87 MG xenograft tumors were given i.p. injections ofsaline (â€¢)or 1 cycle of 5 mg/kg b.i.d. of BE-4-4-4-4 ( 0 ) on days 0-3 and 7-10. B andD, mice were treated with 50 mg/kg BCNU alone on day 4 (D) or BE-4-4-4-4 (sametreatment schedule) plus BCNU 40 mg/kg for 1 cycle (A). A and B. tumor volume (mm1)

for each group. C and D, anima! weights for each group. The doubling time of the tumorfrom 500 to 1000 mm3 was 3 days. Points, the mean tumor volume and animal weight for

8-10 mice per group; bars. SE.

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Fig. 3. Tumor growth rate of A549 xenografts treated with BE-4-4-4-4 Â±BCNU. Aand C, nude mice carrying A549 xenograft tumors were given i.p. injections of saline (â€¢)or 1 cycle of 5 mg/kg b.i.d. of BE-4-4-4-4 ( 0 ) on days 0-3 and 7-10 (cycle 1) or 2 cycles(4) on additional days 42-45 and 49â€”52.B and D, mice were treated with 50 mg/kgBCNU alone on day 4 (D) or BE-4-4-4-4 (same treatment schedule) plus BCNU 40 mg/kgfor 1 cycle (A) or 2 cycles (A) as described above. A and B, tumor volume (mm3) for each

group. C and D, animal weights for each group. The doubling time of the tumor from 500to 1000 mm3 was 26 days. Points, the mean tumor volume and body weight for 3-7 mice

per group; bars, SE.

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Fig. 4. Tumor growth rate of HCT116 xenografts treated with BE-4-4-4-4. Nude micecarrying HCT116 xenograft tumors were given i.p. injections of saline (â€¢)or 5 mg/kgb.i.d. of BE-4-4-4-4 ( 0 ) on days 0-3 and 7-10. A, tumor volume (mm3) for each group.B, animal weights for each group. The doubling time of the tumor from 500 to 1000 mm3

was 12 days. Points, the mean tumor volume and animal weight for four mice per group;bars. SE.

nificant benefit of 1 or 2 cycles of BE-4-4-4-4 treatment in SF-767glioma and A549 lung tumor xenografts. The growth rate of SF-767

tumors slowed after a second cycle, indicating that tumors treated with1 cycle of BE-4-4-4-4 were not resistant to a second and that a second

treatment may be useful in clinical trials. Furthermore, after 2 cycles,3 of 8 mice had complete tumor regressions that lasted for the rest ofthe experiment (76 days). A significant tumor response was alsoobserved for the HCT116 and HT29 tumors. The U-87 MG tumor wasthe least responsive to BE-4-4-4-4, although growth inhibition was

observed.The animal weight loss varied between 4 and 12% at 10-15 days

posttreatment for animals receiving BE-4-4-4-4 alone. Although there

was a significant difference in tumor growth rate, there was not asignificant difference in weight loss on the day of maximum weightloss in animals carrying HT29, A549, and SF767 tumors. For example, on day 14 in HT29 tumor-bearing animals, the difference in

weight was 20.6 Â±1.1 for control and 21.8 Â±0.3 for treated animals(P = 0.31); however, the difference in tumor volume was 794 Â±110for control and 408 Â±49 for treated animals (P = 0.009). This

indicates that transient weight loss does not contribute to the reductionin tumor growth in our models.

The mechanism by which polyamine inhibitors produce an antitu-

mor effect is unclear. One possible mechanism is polyamine depletion(1,2). However, using genetically engineered CHO cells, Ghoda et al.(34) showed that the growth inhibitory effects of specific polyamine

analogues were related to analogue uptake but not to polyaminedepletion. This observation was corroborated by Albanese et al. (35),who found that the growth inhibitory activity of a polyamine analoguewas not due to effects on ODC, polyamine depletion, mitochondrialDNA synthesis, or cellular respiration. Our in vitro and in vivo studieswith A^A^-bistethyOnomospermine and BE-4-4-4-4 in human glioma cell lines (13, 28)4 indicated that polyamine depletion is not

correlated with the growth inhibitory and cytotoxic effects of theseanalogues. Our histopathological data on A549 tumor xenograftsshowed an 18-fold decrease in the mitotic index of BE-4-4-4-4-treated

tumors compared to controls. It is possible that a decrease in mitoticindex by BE-4-4-4-4 correlates with inhibition of cell growth.

A second possible mechanism of action for polyamine analoguesinvolves the induction of the polyamine catabolizing enzyme SSAT(17, 33). A direct correlation between SSAT induction and growthinhibition was observed in human lung and melanoma cells in response to certain polyamine analogues (12, 17, 33). However, thiscorrelation does not always exist for all cell lines and analogues (34).In SF-767 and U-251 MG cell lines, BE-4-4-4-4 showed no signifi

cant effect on SSAT activity (data not shown) but did result in growtharrest in vitro (28)4 and significant tumor growth inhibition (Table I).4

A third possible mechanism of cytotoxicity is the effect of spermineanalogues on DNA condensation. Spermine increases the extent ofDNA condensation in chromatin and isolated nuclei in vitro (21, 23).Some analogues may be ineffective as growth inhibitors because they

3Ã¶

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1000-

500

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Fig. 5. Tumor growth rate of HT29 xenografts treated with BE-4-4-4-4. Nude micecarrying HT29 xenograft tumors were given i.p. injections of saline (â€¢)or 1 cycle of 5mg/kg b.i.d. of BE-4-4-4-4 ( 0 ) on days 0-3 and 7-10. A, tumor volume (mm3) for each

group. B, animal weights for each group. The doubling time of the tumor from 500 to 1000mm3 was 9 days. Points, the mean tumor volume and animal weight for eight mice per

group; bars, SE.

4701




are as efficient as spermine is in condensing DNA and, thereby, maymimic the biological functions of naturally occurring polyamines (22).BE-4-4-4-4 has a greater affinity for DNA and is slightly less effective

than spermine at condensing and aggregating DNA (26). Thus, it mayact as an antiproliferative agent by replacing spermine at binding siteson DNA without fulfilling its biological functions (35). The possibility that intracellular binding sites other than DNA are involved inanalogue cell killing potential must not be overlooked.

The growth inhibitory effects of BCNU alone and in combinationwith BE-4-4-4-4 were compared to effects of BE-4-4-4-4 alone inmice with A549 and U-87 MG tumors. BCNU inhibited the growth ofthe U-87 MG tumors; BE-4-4-4-4 added to the regimen significantly

affected weight loss but not tumor burden. The A549 tumors wereextremely responsive to BE-4-4-4-4; the addition of BCNU improved

tumor growth inhibition slightly.The two brain tumors SF-767 and U-87 MG responded very dif

ferently to treatment with BE-4-4-4-4 alone. Complete regressionswere observed in 3 of 8 mice with SF-767 tumors, but those with U-87

MG tumors responded poorly and would be considered resistant toBE-4-4-4-4. In contrast, studies of a series of seven brain tumor celllines in tissue culture found that SF-767 cells were the most resistantto BE-4-4-4-4, whereas U-87 MG cells were quite sensitive (25).

While we have no good explanation for the discrepancy between ourobservations in xenografts and the in vitro results, the in vitro studiesalso showed that natural polyamines block growth inhibition in SF-763 and U-251 MG cells when given together with BE-4-4-4-4 (28).

The extent of blocking is different for these two cell lines. Because inanimals, the treatment may be carried out in the presence of appreciable serum polyamines, the results of polyamine blocking studiesmay be more therapeutically relevant. We are now beginning studiesof the ability of polyamines to block BE-4-4-4-4 action in U-87 MGand SF-767 cells. The discrepancy between the in vitro and in vivo

results may be better understood when the results of those studies areavailable. Differences in drug uptake, differential distribution of thedrug to different tissues, or yet unknown factor(s) in vivo may also beresponsible for the difference we observed between tissue culture andnude mice.

ACKNOWLEDGMENTS

We thank Dr. Ralph Bunte for expert veterinary pathological evaluation. Wealso thank Pamela Derish for editorial assistance.

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1994;54:4698-4702. Cancer Res M. Eileen Dolan, Matthew J. Fleig, Burt G. Feuerstein, et al. Human Tumor XenograftsEffect of 1,19-Bis(ethylamino)-5,10,15-triazanonadecane on

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