Enzymatic spermine metabolites induce apoptosis in neuroblastoma cells associated with increase of p53, Caspase-3 and miR-34a

Neuroblastoma (NB) is a common malignant solid tumor in children, which originates from the sympathoadrenal lineage of neural crest and accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is a well-established poor prognostic marker for neuroblastoma. Whilst N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment. Therefore, new therapies for patients with N-Myc amplified NB need to be developed. The in situ formation of cytotoxic polyamine metabolites by bovine serum amino oxidase (BSAO) is a recent approach in cancer enzymotherapy. It was demonstrated that BSAO and spermine (SPM) addition to cancer cells induces cell growth inhibition and apoptosis through the oxidative stress caused by polyamine metabolites, H2O2 and aldehydes, produced by the oxidative reaction [1]. The cytotoxic effect induced by BSAO and SPM was evalulated by both a clonogenic and MTT assays. The detection of apoptosis in NB cells was evaluated by flow cytometry after Annexin V-FITC labelling and DNA staining with propidium iodide. The percentages of Annexin V-positive cells matched quite well with that of cells showing hypodiploid sub-G1 peak. An increase in mitochondrial membrane depolarization (MMD) was found in NB cells treated with the enzymatic system. The mitochondrial membrane potential activity was checked by flow cytometry studies, labelling cells with the probe JC-1 dye. We also analysed by real time RT-PCR the transcript of some genes involved in the apoptotic process, to determine possible down- or up-regulation of mRNAs after the treatment on SJNKP and the N-Myc amplified IMR-5 cell lines with BSAO and SPM. The experiments were carried out considering the pro-apoptotic genes TP53 and CASPASE-3. After treatment with BSAO and SPM, both cell lines displayed increased mRNA levels for all these pro-apoptotic genes. Interestingly, the pro-apoptotic Sirt-1 inhibitor microRNA miR-34a are increased in SJNKP and IMR5 cells treated with BSAO and SPM. Western blotting analysis with PARP and Caspase 3 antibody support the concept that BSAO/SPM treatment induces high levels of apoptosis in NB cell lines. In addition, to check if there is any difference between neurons and neuroblastoma cells treated with BSAO/SPM, acridine orange/ethidium bromide staining was used. These results suggested that primary neurons are more resistant to the cytotoxic effects induced by hydrogen peroxide and acrolein than NB cells. The major conclusion is that BSAO/SPM treatment leads to anti-proliferative and cytotoxic activity of both NB cell lines, associated with activation of apoptosis. Moreover, the findings suggested that enzymatic spermine metabolite could be a powerful tool in the development of new anticancer treatments. As a future perspective, first of all, to deliver the enzyme into tumor mass, the enzyme has to be conjugated to biocompatible and biodegradable vehicles. In fact, it is expected that after delivering the enzyme directly into cancer cells, toxic oxidation products can be produced intracellularly from endogenous polyamines. Recently, BSAO is going to be bound to novel superparamagnetic surface-active maghemite nanoparticle (SAMN). These nanoparticles are characterized by specific chemical behavior without any superficial modification or coating derivatization. SAMN-BSAO kept an elevated catalytic activity, but it was lower than that of free BSAO [2]. Additional research is needed to improve SAMN-BSAO