The polyamines spermine, spermidine and putrescine are ubiquitous cell components. If they accumulate excessively within the cells, due either to very high extracellular concentrations or to deregulation of the systems which control polyamine homeostasis, they can induce toxic effects. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper containing amine oxidases. Polyamine concentrations are high in growing tissues such as tumors. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. These enzymes catalyze the oxidative deamination of biogenic amines and polyamines to generate the reaction products H2O2 and aldehyde(s) that are able to induce cell death in several cultured human tumor cell lines. H2O2 generated by the oxidation reaction is able to cross the inner membrane of mitochondria and directly interact with endogenous molecules and structures, inducing an intense oxidative stress. Since amine oxidases are involved in many crucial physiopathological processes, investigations on their involvement in human diseases offer great opportunities to enter novel classes of therapeutic agents.

In situ formation of cytotoxic metabolites by an enzyme-catalyzed reaction is a recent approach in cancer chemotherapy. We demonstrate that multidrug resistant human melanoma cells (M14 ADR) are more sensitive than the corresponding wild type cells (M14 WT) to hydrogen peroxide and aldehydes, the products of bovine serum amine oxidase (BSAO)-catalyzed oxidation of spermine. Hydrogen peroxide was mainly responsible for the loss of cell viability. With about 20%, the aldehydes formed from spermine contribute also to cytotoxicity. Elevation of temperature from 37 degrees C to 42 degrees C decreased survival of both cell lines by about one log unit. Pre-treatment with N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527), a lysosomotropic compound, sensitized cells to toxic spermine metabolites. MDL 72527 (at 300 microM) produced in M14 cells numerous cytoplasmic vacuoles which, however, disappeared by 24 h, even in the presence of the drug. Mitochondrial damage, as observed by transmission electron microscopy, correlated better with the cytotoxic effects of the treatment than vacuole formation. Since the release of lysosomal enzymes causes oxidative stress and apoptosis, we suggest that the lysosomotropic effect of MDL 72527 is the major reason for its sensitizing effect.

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Abstract. Hyperthermia is currently receiving widespread attention when associated with other therapeutic modalities, such as irradiation or chemotherapy, in the treatment of cancer. The occurrence of resistance to cytotoxic pharmacological agents in tumor cells, associated with several phenotypic alterations, is one of the major obstacles to successful anticancer chemotherapy. We investigated a new strategy to overcome multidrug resistance (MDR) cancer cells, using bovine serum amine oxidase (BSAO), which forms toxic products from spermine (H 2 O 2 and aldehydes). The cytotoxicity of the products was evaluated in drug-sensitive (LoVo WT) and multidrug-resistant (LoVo DX) colon adenocarcinoma cells at 37 and 42˚C, using a clonogenic cell survival assay. Cytotoxicity was considerably enhanced at 42˚C. Both toxic species contributed to the thermal enhancement of cytotoxicity induced by BSAO and spermine. Cytotoxicity was eliminated in the presence of catalase and aldehyde dehydrogenase (ALDH). An interesting finding was that BSAO and spermine at <1 μM, which were non toxic at 37˚C, became cytotoxic at 42˚C and resemble thermosensitizers. Cell survival results and electron microscopy investigations suggest that, at 42˚C, LoVo DX cells are not resistant to the cytotoxic enzymatic oxidation products of spermine, as was already demonstrated in these cells at 37˚C. Moreover, microscopy modifications caused by both toxic products were more pronounced in LoVo DX than in LoVo WT cells, where morphological cytoplasmatic alterations were shown. Our findings suggest that hyperthermia combined with the enzymatic toxic oxidation products of spermine might be a promising anticancer strategy, mainly against MDR tumor cells.

Abstract.The in situ formation of cytotoxic metabolites by an enzyme-catalyzed reaction is a recent approach in cancer therapy. The present results show that multidrug-resistant human colon adenocarcinoma cells (LoVo) are significantly more sensitive than corresponding wild-type cells to hydrogen peroxide and aldehydes, the products of bovine serum amine oxidase (BSAO)-catalyzed oxidation of spermine. Pretreatment of the cells with N 1 ,N 4 -bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527), a lysosomotropic compound, sensitized both cell lines to the subsequent exposure to spermine metabolites, as was evident from the decrease of cell survival by a log unit. The sensitizing effect was greater in the case of the multidrug-resistant cell line, an aspect of particular importance with respect to potential therapeutic applications of the method, since conventional cancer therapy suffers from the development of drug resistance. Cell viability was determined using a clonogenic assay. MDL 72527 (at 300 μM) produced numerous cytoplasmic vacuoles, presumably of lysosomal origin, after 6-h exposure, which decreased in size and number (in the presence of the drug) by 24 h and had almost disappeared completely at 48 h. Mitochondrial damage, as observed by transmission electron microscopy, seemed to correlate better with the cytotoxic effects of the treatment than the formation of vacuoles. We suggest that the release of lysosomal enzymes into the cytosol by MDL 72527 is the main reason for its sensitizing effect. It is known that lysosomotropic compounds, which release lysosomal enzymes, produce oxidative stress and apoptosis.