Sensitization of human colon adenocarcinoma cells (LoVo) to reactive oxygen species by a lysosomotropic compound.

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. Pre-treatment of the cells with N1,N4-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 microM) 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.

Toxicity of enzymatic oxidation products of spermine to human melanoma cells (M14): sensitization by heat and MDL 72527.

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.

Hyperthermia enhances cytotoxicity of amine oxidase and spermine on drug-resistant LoVo colon adenocarcinoma cells.

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 (H2O2 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 degrees C, using a clonogenic cell survival assay. Cytotoxicity was considerably enhanced at 42 degrees 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 microM, which were non toxic at 37 degrees C, became cytotoxic at 42 degrees C and resemble thermosensitizers. Cell survival results and electron microscopy investigations suggest that, at 42 degrees C, LoVo DX cells are not resistant to the cytotoxic enzymatic oxidation products of spermine, as was already demonstrated in these cells at 37 degrees 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.

Interaction of bovine serum amine oxidase with the polyamine oxidase inactivator MDL 72527.

MDL 72527 was considered a selective inhibitor of FAD-dependent polyamine oxidases. In the present communication, we demonstrate that MDL 72527 inactivates bovine serum amine oxidase, a copper-containing, TPQ-enzyme, time-dependently at 25 degrees C. In striking contrast, the enzyme remained active after incubation with excessive MDL 72527 at 37 degrees C, even after 70 h of incubation. Inactivation of BSAO with MDL 72527 at 25 degrees C did not involve the cofactor, as was shown by spectroscopy and by reaction with phenylhydrazine. Docking of MDL 72527 is difficult, owing to its size and two lipophilic moieties, and it has been shown that minor changes in reaction rate of substrates cause major changes in K(m) and k(cat)/K(m). We hypothesise that subtle conformational changes between 25 and 37 degrees C impair MDL 72527 from productive binding and prevent the nucleophilic group from reacting with the double bond system.