Neuroblastoma (NB) is the most common cancer in infancy and most frequent cause of death from extracranial solid tumors in children. Therefore, besides the current treatments that include chemotherapy, surgery, and radiation, new therapies need to be developed. The in situ formation of cytotoxic polyamine metabolites by bovine serum amino oxidase (BSAO) is a recent approach in cancer therapy. It was demonstrated that BSAO and spermine (SPM) addition to cancer cells induces cell growth inhibition by apoptosis caused by H2O2 and aldehydes, produced by the oxidative reaction. The induction 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 neuroblastoma cells treated with the enzymatic system. 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 the SJ-N-KP cell line with BSAO and SPM. The experiments were carried out considering the pro-apoptotic genes P53, PUMA and CASPASE-3. After treatment with BSAO and SPM, the SJ-N-KP 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-N-KP cells treated with BSAO and SPM. These data support the concept that BSAO/SPM treatment induces high levels of apoptosis. Previously we demonstrated that multidrug resistant (MDR) colon adenocarcinoma cells (LoVo DX) are more sensitive than the corresponding wild type cells (LoVo WT) to H2O2 and aldehydes. Transmission electron microscopy observations showed evident mitochondria alterations. The mitochondrial activity was checked by flow cytometry studies, labelling cells with the probe JC1. After treatment with BSAO/SPM the cells showed a marked increase in MMD. In order to have more information on the effect of BSAO/SPM in cancer 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 canonical pathways that exhibited the largest differences between BSAO treated and untreated cells in the presence of SPM include mitochondrial dysfunction. We conclude that the mechanism of the cytotoxicity of BSAO/SPM is partly related to mitochondrial dysfunction.

Bovine serum amine oxidase and spermine cause apoptosis in cancer cells detected by flow cytometry, real time RT-PCR and proteomic analyses

Finotti, A
Secondo
;
Gasparello, J;Gambari, R
Ultimo
2018

Abstract

Neuroblastoma (NB) is the most common cancer in infancy and most frequent cause of death from extracranial solid tumors in children. Therefore, besides the current treatments that include chemotherapy, surgery, and radiation, new therapies need to be developed. The in situ formation of cytotoxic polyamine metabolites by bovine serum amino oxidase (BSAO) is a recent approach in cancer therapy. It was demonstrated that BSAO and spermine (SPM) addition to cancer cells induces cell growth inhibition by apoptosis caused by H2O2 and aldehydes, produced by the oxidative reaction. The induction 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 neuroblastoma cells treated with the enzymatic system. 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 the SJ-N-KP cell line with BSAO and SPM. The experiments were carried out considering the pro-apoptotic genes P53, PUMA and CASPASE-3. After treatment with BSAO and SPM, the SJ-N-KP 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-N-KP cells treated with BSAO and SPM. These data support the concept that BSAO/SPM treatment induces high levels of apoptosis. Previously we demonstrated that multidrug resistant (MDR) colon adenocarcinoma cells (LoVo DX) are more sensitive than the corresponding wild type cells (LoVo WT) to H2O2 and aldehydes. Transmission electron microscopy observations showed evident mitochondria alterations. The mitochondrial activity was checked by flow cytometry studies, labelling cells with the probe JC1. After treatment with BSAO/SPM the cells showed a marked increase in MMD. In order to have more information on the effect of BSAO/SPM in cancer 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 canonical pathways that exhibited the largest differences between BSAO treated and untreated cells in the presence of SPM include mitochondrial dysfunction. We conclude that the mechanism of the cytotoxicity of BSAO/SPM is partly related to mitochondrial dysfunction.
2018
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2413763
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