Cytotoxic products of polyamines generated in situ by an enzyme-catalysed reaction may be useful as a new avenue in combating cancer. This study demonstrated that MDR (multidrug-resistant) cancer cells (colon adenocarcinoma and melanoma) are significantly more sensitive than the corresponding WT (wild-type) ones to H2O2 and aldehydes, the products of BSAO (bovine serum amine oxidase)-catalysed oxidation of spermine. Moreover, cytotoxicity was considerably greater when the treatment was carried out at 42°C than at 37°C. TEM (transmission electron microscopy) observations showed major ultrastructural alterations of the mitochondria. These were more pronounced in MDR than in WT cells. After treatment with BSAO/spermine, a higher mitochondrial membrane depolarization and an increased mitochondrial activity in drug-resistant cells were observed.
The cytotoxicity of BSAO (bovine serum amine oxidase) (EC 184.108.40.206)/spermine was potentiated by the pre-treatment of the cells with the polyamine oxidase inhibitor MDL 72527 [N1,N4-bis(2,3-butadienyl)-1,4-butanediamine], a lysosomotropic compound. Pre-treatment for several exposure times to 300 μM MDL 72527 sensitized both WT (wild-type) and MDR (multidrug-resistant) melanoma and adenocarcinoma cell lines to the subsequent treatment with spermine metabolites. Cytotoxicity was greater by the combined treatment than by BSAO/spermine alone, even though MDL 72527 did not reduce the number of viable cells under our experimental conditions. Pre-treatment with MDL 72527 induced the formation within 6 h of numerous cytoplasmic vacuoles of lysosomal origin. These vacuoles decreased at 24 h and disappeared nearly completely at 48 h. Mitochondrial damage, as observed by TEM (transmission electron microscopy), seemed to correlate better with the cytotoxic effects of the treatment than the formation of vacuoles. MDL 72527 induced the release of Acridine Orange from the lysosomes into the cytoplasm. It is known that the release of lysosomal enzymes produces oxidative stress and apoptosis. This might be the main reason for the sensitizing effect of MDL 72527. The results suggest that enzymatically formed cytotoxic agents, alone or in association with lysosomotropic compounds or heat treatment, activate stress-activated signal transduction pathways, leading to apoptotic cell death, mainly in MDR cell lines.
The polyamines spermine, spermidine and putrescine are ubiquitous cell components. If they accumulate excessively within the cells, either due to very high extracellular amounts or to deregulation of the systems which control polyamine homoeostasis, they can induce toxic effects. These molecules are substrates of a family of enzymes, the amine oxidases, that includes copper-containing amine oxidases isolated from serum . These enzymes are important because they contribute to the regulation of levels of mono- and poly-amines. Amine oxidases catalyse the oxidative deamination of polyamines to generate the reaction products H2O2 and aldehyde(s) . Such toxic products are able to induce stress-activated signal transduction pathways, leading to cell death, necrosis or apoptosis, in several tumour-cultured cell lines [2–4]. The difference between normal and tumour cells is related to polyamine content and metabolism. Polyamine concentrations are high in growing tissues such as tumours, for example, in breast and colon cancer .
Cytotoxic products of spermine formed in situ by an enzyme-catalysed reaction might be useful for the destruction of tumours. Therefore this research explores the possibility of using purified BSAO in the presence of exogenous spermine or endogenous polyamines, after injection of the enzyme into the tumour, to induce cytotoxicity . BSAO is a copper-containing glycoprotein of molecular mass 170 kDa, which oxidatively deaminates the primary amino groups of polyamines, such as spermine and spermidine. The reaction involves dioxygen and water as substrates . The products are H2O2, aldehydes and ammonia . In the case of spermine, the monoaldehyde, the unstable dialdehyde, and a further breakdown product, which is likely to be acrolein, may be formed [9,10] (1).
Reaction for spermine oxidation in the presence of BSAO.
In this study, the mechanism of cell death of drug-sensitive and MDR human cancer cells, induced by the enzymatic toxic products, was investigated.
The development of drug-resistant tumour cells, following exposure to cytotoxic drugs, is a major obstacle of conventional anticancer chemotherapy. Multidrug resistance is associated with phenotypic alterations. MDR cancer cells usually display a decrease in intracellular drug accumulation and/or drug distribution due to the overexpression of genes which encode membrane-bound transporter proteins, such as 170 kDa P-gp (P-glycoprotein) [11,12]. P-gp functions as an energy-dependent pump, which expels drugs out of cells .
According to previous observations on P-gp-overexpressing MDR CHO (Chinese-hamster ovary) cells , our results demonstrate that MDR human cancer cells are significantly more sensitive than the corresponding WT ones to H2O2 and aldehyde(s), the products of BSAO-catalysed oxidation of spermine, suggesting a possible new strategy against MDR tumours.
The cytotoxicity induced by the enzymatic oxidation products of polyamines was evaluated on both DX (doxorubicin)-sensitive and -resistant human colon adenocarcinoma cells (LoVo WT and LoVo DX respectively) by plating efficiency assay, and the ultrastructural alterations were investigated by TEM.
Figure 1 shows the percentage cell survival against the time of exposure to purified BSAO (6.98×10−3 units/ml) in the presence of exogenous spermine (6 and 12 μM), with and without the catalase alone or catalase and ALDH (aldehyde dehydrogenase), at 37°C. In the presence of BSAO and spermine alone, a higher cytotoxicity was observed in LoVo DX than in LoVo WT cells. The percentage cell survival decreased in both cell lines with increasing exposure time, resulting in approx. 18% in LoVo WT (Figure 1, curve a) and approx. 5% in LoVo DX cells (Figure 1, curve b), after 60 min of incubation. As expected, it may be seen that the cytotoxic effect in LoVo WT (Figure 1, curve c) and LoVo DX cells (Figure 1, curve d) was more marked in the presence of 12 μM spermine than 6 μM spermine, at 37°C. The higher cytotoxicity in the presence of 12 μM spermine was attributed to the formation of an increased amount of H2O2 and aldehyde(s) during the enzymatic reaction.
Effect of catalase and ALDH on cytotoxicity induced by BSAO in the presence of spermine
In order to evaluate the contribution of each enzymatic oxidation product in the inhibition of cell growth, experiments were performed in the presence of exogenous catalase, an enzyme that decomposes H2O2, or catalase and ALDH added simultaneously to the incubation mixture. Catalase (240 units/ml) afforded a marked reduction of the cytotoxic effect, corresponding to approx. 80% cell survival, on LoVo WT and LoVo DX cells (Figure 1, curves e and f respectively), probably due to the clearance of H2O2, formed in the catalytic reaction by the enzyme. The result showed that H2O2 was not the sole toxic factor and that other products of the enzymatic oxidative deamination were involved, such as aldehyde(s), including acrolein spontaneously formed from the aminoaldehydes . The addition of exogenous NAD-dependent ALDH (0.4 unit/ml) metabolized the aldehyde to form the corresponding carboxylic acid and prevented the toxic effects of acrolein. In fact, after addition of both exogenous enzymes, catalase and NAD-dependent ALDH, cytotoxicity was completely inhibited throughout the 60 min of incubation (Figure 1, curves g and h).
In order to reveal the intracellular targets involved in the mechanisms responsible for the higher cytotoxic effect of the enzymatic products, H2O2 and aldehyde(s), on the drugresistant rather than sensitive cells, TEM observations and cytofluorimetric analysis were carried out. These studies revealed major pathological modifications of the mitochondria.
Both control LoVo WT (Figure 2a) and LoVo DX cells (Figure 2b) showed a well-preserved ultrastructure when observed by TEM. The nucleus was clearly defined with dispersed chromatin, while the cytoplasm was characterized by the presence of mitochondria with parallel cristae in a dense and uniform matrix. After treatment (BSAO and 6 μM spermine), LoVo WT cells did not show any consistent ultrastructural alteration (Figure 2c). In contrast, MDR cells exhibited marked modifications; in particular, all mitochondria showed a highly condensed matrix and dilated cristae (Figure 2d). No increase in the overall size of the mitochondria was observed. The treatment with BSAO or spermine alone did not induce any detectable alteration in the ultrastructure of either cell line.
Transmission electron microscopy observations
Mitochondria therefore appear to play a pivotal role in determining the differential response between sensitive and drug-resistant cells.
The higher sensitivity to cytotoxic spermine derivatives observed in LoVo DX cells compared with their sensitive counterparts was not related to a different content of glutathione, as observed previously on CHO (CHRC5) cells resistant to colchicine . LoVo cells have the same glutathione pool and in colon adenocarcinoma LoVo DX cells this phenomenon has been attributed to an earlier and higher mitochondrial membrane depolarization. Moreover, a higher basal production of ROS (reactive oxygen species) in MDR cells than in the drug-sensitive cells was detected, suggesting an increased mitochondrial electron-transport chain activity in drug-resistant cells [3,15].
Our study also deals with the possible effects of both H2O2 and aldehyde (produced by the BSAO/polyamine-spermine enzymatic system) in inducing considerably greater cytotoxicity at 42°C than at 37°C . Regional hyperthermia potentiates the cytotoxic action of many different anticancer drugs and has considerable potential in cancer treatment [17,18]. Promising results are emerging from clinical studies involving hyperthermia combined with chemotherapy . In fact, a biological strategy to enhance the therapeutic effects of hyperthermia is to use heat together with pharmacological agents that become much more cytotoxic at high temperatures. These compounds, such as cysteamine and aminothiol N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065), defined as thermosensitizers, are not toxic at 37°C, but at elevated temperatures they become potent cell inactivators . Another group of drugs, all of which were considered to be heat sensitizers, are the naturally occurring polyamines putrescine, spermine and spermidine .
The enzymatic oxidation products of spermine behaved similarly to other thermosensitizers, such as aminothiol WR-1065 or cysteamine [22,23]. Beneficial effects could therefore be achieved using localized heating to enhance the action of toxic products generated by BSAO and spermine within the tumour region, without increasing normal tissue damage. An interesting finding was observed in human colon adenocarcinoma and melanoma cells when conditions of BSAO and spermine levels of less than 1 μM that were non-toxic at 37°C became cytotoxic at 42°C and resemble thermosensitizers . These results suggest that the combination of hyperthermia and spermine enzymatic oxidation products could prove to be useful in cancer treatment and mainly effective against MDR cells.
To take advantage of the higher levels of polyamines in tumour compared with normal tissues, as reported previously , toxic products such as H2O2 and aldehyde(s) could be generated in situ by delivering amine oxidases into tumours to induce cell death [14,24]. For cancer therapies, it is also important to establish the mechanism(s) by which cytotoxic agents cause tumour cell death. Apoptosis is a highly regulated form of cell death involving many different genes and proteins . During apoptosis, caspase enzymes are activated, and chromatin condensation and internucleosomal degradation of DNA occur in the nucleus. The apoptotic process avoids liberation of cellular contents into the surrounding tissue to prevent induction of inflammation, as occurs when cells lose membrane integrity and die by necrosis.
It has been therefore evaluated in vivo, using a mouse melanoma model, whether BSAO, when injected directly into the tumour, is able to induce tumoricidal activity by converting endogenous polyamines into toxic products in situ. It has been established previously that immobilization of enzymes such as asparaginases into polymeric matrices such as PEG [poly(ethylene glycol)] increases their structural stability and functional activities in vitro and in vivo . Immobilization of BSAO into a biocompatible matrix, made of BSA and a PEG, was reported , and the enzyme showed a higher operational stability relatively to its native form. Therefore both native and immobilized BSAO were compared in vivo in terms of their respective abilities to induce on mice melanomas cell death by either apoptosis or necrosis .
When immobilized BSAO was injected into the tumour, there was a marked decrease (70%) of the tumour growth. This was compared with a decrease of only 32% in tumour size when the same amount of native BSAO was administered. The mechanism of tumour cell death was also determined. When tumours were treated with immobilized BSAO, there was induction of a high level of apoptosis (approx. 70%), compared with less than 10% with the native enzyme. Native BSAO, probably due to a burst of cytotoxic products, induced a high level of necrosis of approx. 40%, compared with less than 10% with immobilized BSAO. The advantage is that immobilized BSAO can act by allowing the slow release of cytotoxic products, which induces tumour cell death by apoptosis rather than necrosis.
Currently, we are studying drug combinations with the aim of increasing the induction of cell death by toxic polyamine metabolites. The cytotoxicity of BSAO/spermine was enhanced by pre-treatment of the cells with MDL 72527. This compound is an inactivator of FAD-dependent polyamine oxidase and represents a lysosomotropic compound  that has been demonstrated previously to improve the anti-tumour effect of DFMO (difluoromethylornithine) . At present, DFMO is undergoing clinical evaluation as a chemoprevention agent . MDL 72527 has cytotoxic properties which, however, are unrelated to its ability to inactivate polyamine oxidase .
In the present study, emphasis was put on the ability of MDL 72527 to sensitize LoVo cells to H2O2 and aldehyde generated from BSAO/spermine-induced cell death .
Cell-survival experiments were performed on human adenocarcinoma and melanoma cells (M14) . Pre-treatment with 300 μM MDL 72527 for 6, 24 or 48 h sensitized both WT and MDR adenocarcinoma and melanoma cell lines to the subsequent exposure to spermine metabolites. An obvious time-dependence was observed for optimum sensitization. The lowest cell survival was observed for 24 h of pre-incubation with MDL 72527. Cytotoxicity was significantly greater by the combined treatment than by BSAO/spermine alone, even though MDL 72527 alone did not reduce the number of viable cells under the experimental conditions. An impairment of cell metabolism by this drug was, however, indicated by the formation within 6 h of numerous cytoplasmic vacuoles of lysosomal origin. The number and size of vacuoles decreased at 24 h and disappeared nearly completely at 48 h. After this time, the cells were able to recover physiological functions.
Mitochondrial damage, as observed by TEM, seemed to correlate better with the cytotoxic effects of the treatment, than the formation of vacuoles. These vacuoles are most probably of lysosomal origin. A diamine, structurally related to MDL 72527 (2,5-diamino-3-hexene)  and the known lysosomotropic compound chloroquine cause the formation of very similar vacuoles in leukaemic cells . In contrast with the vacuoles in leukaemic cell lines, the vacuoles in LoVo cells did not coalesce to form larger vacuoles with time, but, on the contrary, they disappeared nearly completely in the presence of MDL 72527 at 48 h of incubation. However, vacuole formation did not correlate directly with the loss of cell viability.
Instead, the results support the hypothesis that the release of lysosomal enzymes into the cytosol by MDL 72527 is the major reason for its sensitizing effect. The findings demonstrate that the lysosomotropic effect of MDL 72527 is maximal within less than 24 h, while at longer times compensatory reactions allow cells to recover physiological function to some extent. It is known that oxidative stress stimulates defence mechanisms by inducing enzymes which assist in antagonizing oxidative damage . Experiments with Acridine Orange-stained cells support this view. MDL 72527 releases Acridine Orange from the lysosomes into the cytoplasm. It is known that the release of lysosomal enzymes produces oxidative stress and that an important role of lysosomes in both necrotic and apoptotic cell death is well founded .
Regarding the impairment of cell viability, the mitochondrial ultrastructural alterations of the cells treated with BSAO/spermine, after pre-treatment with MDL 72527, seem more relevant than the formation of cytoplasmic vacuoles, since they were more accentuated in MDR cells than in the WT ones. Moreover, they seem to be paralleled by the loss of cell viability. It is known from previous work , as reported above, that one of the earliest signs of cell damage by BSAO and spermine is the depolarization of the mitochondrial membranes.
In conclusion, owing to its lysosomotropic effect, pre-treatment with MDL 72527 amplifies the ability of the metabolites formed from spermine by oxidative deamination to induce cell death.
In fact, the results suggest that enzymatically formed cytotoxic agents alone, or in association with either the polyamine oxidase inactivator (MDL 72527) or heat treatment, activate stress signal transduction pathways, leading to apoptotic cell death. We suggest that lysosomotropic compounds are of interest as anticancer agents, mainly in the treatment of MDR tumours.
Health Implications of Dietary Amines: A joint COST Action 922 and Biochemical Society Focused Meeting held at Medico-Chirurgical Hall, University of Aberdeen, U.K., 19–21 October 2006. Organized and Edited by H.M. Wallace (Aberdeen, U.K.).
The original work here reported was partially supported by the Italian MIUR (Ministero dell'Istruzione, dell'Università e della Ricerca) and by funds MIUR-PRIN 2005 (Cofin) (E.A.).