Polyamine metabolites induce apoptosis in cancer cells detected by real time RT-PCR and mass spectroscopy analyses

In situ formation of cytotoxic polyamine metabolites by an enzyme-catalyzed reaction is a recent approach in cancer chemotherapy. We demonstrate that multidrug resistant (MDR) colon adenocarcinoma cells (LoVo DX) are more sensitive than the corresponding wild type cells (LoVo WT) to H 2 O 2 and aldehydes, the products of bovine serum amine oxidase (BSAO)-catalyzed oxidation of spermine (SPM). Both cytotoxic metabolites were responsible for the loss of cell viability. Transmission electron microscopy observations showed that BSAO and SPM induced evident mitochondria alterations, more pronounced in LoVo DX than in LoVo WT cells. The mitochondrial activity was checked by flow cytometry studies, labelling cells with the probe JC1, that displayed a basal hyperpolarized status of the mitochondria in LoVo DX cells. After treatment with BSAO and SPM, the cells showed a marked increase in mitochondrial membrane depolarization (MMD), higher in LoVo DX than in LoVo WT cells. An increasing in MMD was also found in neuroblastoma SJ-NKP cell line treated with BSAO and 18 µM SPM concentration. We 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 of SJ- NKP cell line with BSAO and SPM. Experiments were carried out considering the pro- apoptotic genes P53, PUMA and CASPASE-3. After treatment with BSAO and SPM, the SJ-NKP cells displayed increased mRNA levels for all these pro-apoptotic genes. Interestingly, the pro-apoptotic Sirt-1 inhibitor microRNA miR-34a significantly increases in SJ-NKP cells treated with BSAO and SPM. These data support the concept that BSAO/SPM treatment induces high levels of apoptosis. In order to have more information on the effect of BSAO and SPM in tumor cells, a proteomics approach was performed using LoVo cells and prostate cancer (LNCaP) cells treated with and without BSAO/SPM. In total 721 unique proteins were identified in LNCaP cells (more than 4700 in LoVo cells) of which 40 were differentially expressed by more than 1.3 folds. The diseases and biofunctions analyses in the heat map show  increase in cell death of tumor cells. The canonical pathways that exhibited the largest differences between BSAO treated and untreated cells in the presence of SPM include both mitochondrial dysfunction and eIF-2 signaling. Therefore, we conclude that the mechanism of the cytotoxicity of BSAO/SPM is partly related to mitochondrial dysfunction.