Antitumor effects of bis(ethyl)polyamine analogs on mammary tumor development in FVB/NTgN (MMTVneu) transgenic mice
Introduction
The natural polyamines, putrescine (H2N(CH2)4NH2), spermidine (H2N(CH2)3NH(CH2)4NH2), and spermine (H2N(CH2)3NH(CH2)4NH(CH2)3NH2), are intracellular organic cations that are involved in cell proliferation and differentiation [1], [2], [3]. Polyamine levels are tightly regulated by several biosynthetic and metabolic enzymes, including ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC) and spermidine/spermine acetyl transferase (SSAT) which are regulated by trophic stimuli, hormones and growth factors. In addition, polyamines themselves play an important role in the regulation of these enzymes and maintaining polyamine homeostasis [3]. However, polyamine biosynthetic activity and polyamine levels are significantly higher in several tumors, including breast tumors, compared with that of surrounding normal tissues [4], [5], [6]. Elevated polyamine levels appear to play a major role in the development and/or maintenance of neoplastic phenotype [7], [8]. Use of ODC/SAMDC inhibitors has shown polyamine biosynthesis to be an obligatory step in the initiation and maintenance of cell proliferation, suggesting the importance of regulators of polyamine pathway as cancer therapeutic agents [4], [9], [10].
Methylglyoxalbis(guanylhydrazone) (MGBG) and dl-α-difluoromethylornithine (DFMO) are two early agents developed to inhibit SAMDC and ODC, respectively. The success of these agents has been limited due to regulatory homeostatic responses of the polyamine pathway, and the ability of tumor cells to salvage exogenous polyamines from the environment [11], [12]. In order to circumvent this difficulty, polyamine homologs and bis(ethyl)polyamine analogs have been synthesized and tested in cell culture and nude mice xenograft models as they introduce fewer compensatory responses in the polyamine pathway [1], [13]. It is hypothesized that these analogs utilize the polyamine transport pathway and help to maintain the cationic environment. However, they are unable to participate in the regulatory biochemical reactions because of the structural specificity of natural polyamines. In many cases, analogs are known to suppress the activity of ODC and SAMDC, and up-regulate the function of SSAT [13], [14], [15].
Bis(ethyl)polyamine analogs have been shown to suppress tumor growth in several nude mouse xenograft models, including human melanoma [14], [15], human pancreatic adenocarcinoma [16], human colonic tumor [17] and prostate cancer [18]. However, studies on the effect of these analogs on breast cancer models are limited. We therefore undertook this investigation using the FVB/NTgN (MMTVneu) transgenic mouse model. The human homolog of neu oncogene (HER-2/cerbB2) has been shown to be amplified and expressed in a subset of human primary breast cancer, and its overexpression is inversely correlated with patient survival [19], [20], [21], [22]. It has been shown that increased polyamine biosynthetic activity critically interacts with HER2/neu in promoting human mammary cell transformation in culture [23]. FVB/NTgN (MMTVneu) transgenic mice begin to develop mammary tumors at about 4 months of age, with a median incidence of 6.8 months [24], [25]. In the present study, we tested the effects of bis(ethyl)norspermine (C2H5HN(CH2)3NH(CH2)3NH(CH2)3NHC2H5 or BE333) and 1,15bis(ethylamino)4,8,12triazapentadecane (C2H5HN(CH2)3NH(CH2)3NH(CH2)3NH(CH2)3NHC2H5 or BE3333), on these transgenic mice. We found a decrease in mortality and tumor number and volume in treated mice compared to controls. The analog treatment also reduced intracellular polyamine levels in tumor tissues of treated animals and up-regulated the activity of the polyamine catabolizing enzyme, SSAT.
Section snippets
Drug treatment and measurement of tumor growth
BE333 and BE3333 were synthesized as described previously [17]. Analogs were dissolved in sterile saline. FVB/NTgN (MMTVneu) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 4–6 weeks of age. Mice were randomized and divided into control and experimental groups. Mice were injected i.p. with either BE333 (n=8) or BE3333 (n=10) (20 mg/kg body wt) once a week for 14 weeks, starting at 31 weeks of age. We selected this dose in order to minimize toxicity and increase the potential
Effect of bis(ethyl)polyamines on life span
Fig. 1 illustrates the effect of polyamine analog treatment on mortality of the FVB/NTgN transgenic mice. At 44 weeks of age, 90% of treated mice were alive compared with 65% in the control group. However, this reduction was not statistically significant due to the relatively small number of animals in each group. There was no difference in the body weights of mice between the different groups during the course of the treatment. The average weights of mice during the 14 week study period were
Discussion
This investigation shows that the polyamine analogs, BE333 and BE3333 are capable of inhibiting mammary tumor development in FVB/NTgN (MMTVneu) transgenic mice, an animal model of breast cancer. Treatment with these analogs prolonged the life span of these mice, indicating the therapeutic potential of the analogs at a low dose (20 mg/kg body wt), once a week. Treated mice had lower polyamine levels in both tumor and kidney tissues, and SSAT appeared to act as an effector of analog action as its
Acknowledgements
This work was supported, in part, by research grants CA R01 73058 and R01 CA 42439 from the National Cancer Institute (T.T. and T.J.T.), a Collaborative Research Award from the Cancer Institute of New Jersey (T.J.T. and P.A.) and a Grant-in-Aid from the Ministry of Education, Science and Culture (A.S.), Japan.
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