Spermine oxidase (SMO) activity in breast tumor tissues and biochemical analysis of the anticancer spermine analogues BENSpm and CPENSpm

Abstract

Background: Polyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates the expression of spermine oxidase (SMO) and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme.

Methods: BC tissue samples were analyzed for SMO transcript level and SMO activity. Student's t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined.

Results: Both the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors.

Conclusions: This study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials.

Figure 1

RT-PCRs determination of selected genes in BC tissues. Samples from tumor (T) and nontumor (NT) tissues were treated and analyzed as described in Methods. The PCR products were fractionated by 1.2% agarose gel electrophoresis. (A) Representative RT-PCR experiments from three independent replicas are shown. (B) Densitometric analyses of PCR gel bands, obtained from patients, represent the measurements done on three separate experiments. Data were pooled and analyzed to produce an average level of expression. Alternatively, gene of interest/β-actin or/GADPH ratios (normalized for each experimental time point) have been used for normalization. An arbitrary densitometric unit bar graph (SD) is shown. The p values (< 0.01) were measured with the Student's t test.

Figure 2

SMO, APAO, ODC and SSAT activities. Enzyme activities from tumor (T) and nontumor (NT) samples were assayed as described in Methods section. Results are mean (SD) with n value of 20. The p values (< 0.05) were measured with Wilcoxon matched pairs signed rank test.

Figure 3

Schematic representation of the putative complexes formed by mSMO with the substrate Spm (A), the inhibitor MDL 72527 (B), and the analogues BENSpm (C) and CPENSpm (D). For details see Methods section.

Figure 4

Chemical structures of SMO substrates and inhibitors. Chemical structures of Spm, the specific substrate of SMO enzyme, of MDL 72527 (N¹, N⁴-bis(2,3-butadienyl)-1,4butanediamine), an inhibitor of SMO, and of Spm-derivative analogues BENSpm (N¹, N¹¹-di(ethyl)norspermine) and CPENSpm (N¹-cyclopropyl-methyl-N¹¹-ethyl-norspermine). Abbreviations used in the figure are defined in the text.