Spermine oxidase (SMO) is a FAD-containing enzyme while bovine serum amine-oxidase (BSAO) is a copper amine oxidase and both are able to oxidize polyamines. They play a dominant role in the highly regulated mammalian polyamines catabolism. Spermine oxidase specifically oxidizes spermine (SPM) producing spermidine (SPD), the reactive oxygen species H2O2 and the aldehyde 3-aminopropanal, each with the potential to produce cellular damages and pathologies (1). BSAO deaminates SPM and SPD to H2O2, aldehyde acrolein and ammonia. Since SPM represents a common substrate for SMO and BSAO enzymatic activities, a comparative study was performed on both enzymes in presence of SPM on several tumor cell lines melanoma (M14) and colon adenocarcinoma (LoVo) wild type and their MDR counterparts. SPM is a tetramine that plays mandatory roles in several cell functions, such as DNA synthesis, cellular proliferation, modulation of ion channels function and cellular signaling (1). Our previous studies demonstrated that BSAO and SPM added to human cancer cells induce cytotoxicity and overcome the MDR phenotype (2). We have performed in vitro experiments treating the above human cancer cell lines both phenotype wild type and MDR with SMO in presence of several concentrations of SPM (0-80 μM) for 60 min at 37°C. Cytotoxicity induced by SPM oxidation metabolites, such as H2O2 and the aldehyde 3-aminopropanal, was greater in MDR cells than in the corresponding wild-type ones (WT), maybe due to an increased mitochondrial activity. To get structural information a comparative study was also carried out on both enzymes, by differential Scanning Calorimetry (DSC) (3). The thermal stability of BSAO and SMO is studied by differential scanning calorimetry with a MicroCal MC-2D instrument. Differential scanning calorimetry permits detection of overall changes in protein structure correlated with changes in thermal stability of one or more calorimetric domains. This is particularly important in the case of BSAO and SMO, proteins for which only a little structural information is available. Our previous studies demonstrated that the thermal denaturation profile of the dimeric BSAO is characterized by three distinct endothermic peaks (Tm 58°C, 71.3°C, 77.9°C) (3). The deconvolution of the thermal profile required five two-state transitions, revealed for the dimeric protein a five domain structure, while the monomeric SMO displays one single, endothermic peak (Tm 63°C). The peak may be deconvoluted into two transitions and reveals the presents of two non similar size calorimetric domains: the lowest temperature domain is described by a non two-state transition and the highest temperature domain is described by a two-state transition. The thermal denaturation is irreversible after heating to 100°C. Since the treatments with BSAO/SPM and SMO/SPM increased the amount of reactive oxygen species (ROS) inside the cells, higher in MDR than WT ones, we hypothesize that the enhancement of amine oxidase activity in tissues, undergoing pathological proliferative phenomena, may reasonably be exploited in the treatment of neoplastic diseases, mainly against MDR tumors. References: 1- Cervelli M, Amendola R, Polticelli F, Mariottini P (2012). Spermine oxidase ten years later , AminoAcids 42 :441-450 2-Agostinelli, E., Condello, M., Molinari, A., Tempera, G., Viceconte, N., Arancia, G. (2009) Cytotoxicity of spermine oxidation products to multidrug resistant melanoma M14 ADR2 cells: Sensitization by the MDL 72527 lysosomotropic compound International Journal of Oncology 35: 485-498 3) Giartosio A, Agostinelli E and Mondovì B (1988). Domains in bovine serum amine oxidase. Biochem. Biophys. Res. Comm. 154: 66-72.
Cervoni, L., Coccia, M., Meringolo, M., Tempera, G., Cervelli, M., Angelucci, E., et al. (2012). A comparative study between spermine oxidase and bovine serum amine oxidase in differential scanning calorimetry and on cytotoxicity on cancer cell lines. In International Congress on Polyamines. Polyamines: Biological and Clinical Perspectives (pp.205 P26).
A comparative study between spermine oxidase and bovine serum amine oxidase in differential scanning calorimetry and on cytotoxicity on cancer cell lines
CERVELLI, MANUELA;MARIOTTINI, Paolo;
2012-01-01
Abstract
Spermine oxidase (SMO) is a FAD-containing enzyme while bovine serum amine-oxidase (BSAO) is a copper amine oxidase and both are able to oxidize polyamines. They play a dominant role in the highly regulated mammalian polyamines catabolism. Spermine oxidase specifically oxidizes spermine (SPM) producing spermidine (SPD), the reactive oxygen species H2O2 and the aldehyde 3-aminopropanal, each with the potential to produce cellular damages and pathologies (1). BSAO deaminates SPM and SPD to H2O2, aldehyde acrolein and ammonia. Since SPM represents a common substrate for SMO and BSAO enzymatic activities, a comparative study was performed on both enzymes in presence of SPM on several tumor cell lines melanoma (M14) and colon adenocarcinoma (LoVo) wild type and their MDR counterparts. SPM is a tetramine that plays mandatory roles in several cell functions, such as DNA synthesis, cellular proliferation, modulation of ion channels function and cellular signaling (1). Our previous studies demonstrated that BSAO and SPM added to human cancer cells induce cytotoxicity and overcome the MDR phenotype (2). We have performed in vitro experiments treating the above human cancer cell lines both phenotype wild type and MDR with SMO in presence of several concentrations of SPM (0-80 μM) for 60 min at 37°C. Cytotoxicity induced by SPM oxidation metabolites, such as H2O2 and the aldehyde 3-aminopropanal, was greater in MDR cells than in the corresponding wild-type ones (WT), maybe due to an increased mitochondrial activity. To get structural information a comparative study was also carried out on both enzymes, by differential Scanning Calorimetry (DSC) (3). The thermal stability of BSAO and SMO is studied by differential scanning calorimetry with a MicroCal MC-2D instrument. Differential scanning calorimetry permits detection of overall changes in protein structure correlated with changes in thermal stability of one or more calorimetric domains. This is particularly important in the case of BSAO and SMO, proteins for which only a little structural information is available. Our previous studies demonstrated that the thermal denaturation profile of the dimeric BSAO is characterized by three distinct endothermic peaks (Tm 58°C, 71.3°C, 77.9°C) (3). The deconvolution of the thermal profile required five two-state transitions, revealed for the dimeric protein a five domain structure, while the monomeric SMO displays one single, endothermic peak (Tm 63°C). The peak may be deconvoluted into two transitions and reveals the presents of two non similar size calorimetric domains: the lowest temperature domain is described by a non two-state transition and the highest temperature domain is described by a two-state transition. The thermal denaturation is irreversible after heating to 100°C. Since the treatments with BSAO/SPM and SMO/SPM increased the amount of reactive oxygen species (ROS) inside the cells, higher in MDR than WT ones, we hypothesize that the enhancement of amine oxidase activity in tissues, undergoing pathological proliferative phenomena, may reasonably be exploited in the treatment of neoplastic diseases, mainly against MDR tumors. References: 1- Cervelli M, Amendola R, Polticelli F, Mariottini P (2012). Spermine oxidase ten years later , AminoAcids 42 :441-450 2-Agostinelli, E., Condello, M., Molinari, A., Tempera, G., Viceconte, N., Arancia, G. (2009) Cytotoxicity of spermine oxidation products to multidrug resistant melanoma M14 ADR2 cells: Sensitization by the MDL 72527 lysosomotropic compound International Journal of Oncology 35: 485-498 3) Giartosio A, Agostinelli E and Mondovì B (1988). Domains in bovine serum amine oxidase. Biochem. Biophys. Res. Comm. 154: 66-72.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
