Studies of cytotoxicity on human tumor cells induced by enzymatic oxidation products of polyamines: a new approach in cancer therapy

Background The polyamines spermine, spermidine and putrescine are ubiquitous cell components and required for several physiological functions, such as DNA synthesis, cell growth and differentiation. Polyamine concentrations are high in growing tissues such as tumors. Amine oxidases are important because they contribute to the regulation of polyamines’ levels. These enzymes catalyze the oxidative deamination of polyamines to generate the reaction products H2O2 and aldehyde(s), that are able to induce oxidative stress in several cultured human tumor cell lines. Because of the limited success of chemotherapy for tumors, most investigations have oriented on cellular and molecular mechanisms responsible for the onset of multidrug resistance (MDR). Several studies have therefore been performed to overcome the MDR phenotype and to develop innovative chemotherapeutic strategies effective against MDR tumors. Our previous studies using bovine serum amine oxidase (BSAO) have shown that H2O2 and aldehyde(s) were responsible for the cytotoxicity induced in human colon adenocarcinoma cells (LoVo). The treatment of tumors with BSAO could be useful as a new strategy for cancer therapy. In this study, maize polyamine oxidase (ZmPAO), a flavin adenine dinucleotide (FAD)-dependent enzyme, was used due to both its high enzymatic specific activity and the low molecular weight, which are important aspects for the delivery of these enzymes into cancer cells. I investigated on the cytotoxicity and the mechanism of cell death induced by enzymatic oxidation product of polyamine generated by ZmPAO. Methods Subconfluent LoVo WT cells, its doxorubicin resistance (MDR) cells, and AGS gastric tumor cells were used to investigate the cytotoxic effect induced by polyamine metabolites. The harvested tumor cells were treated with exogenous spermine and ZmPAO purified from maize shoot at 37°C up to 1 hr. The treated cells were used in the following assays. Cell viability assays were performed by clonogenic assay. After the treatment, cells were incubated at 37°C for 10-17 days until colonies were formed. The colonies were stained and counted. The involvement of apoptotic cell death was assessed by Annexin V-FITC/PI staining and cell cycle, using flow cytometry. An increasing of the sub-G1 peak was detected. In order to determine the involvement of mitochondria in apoptosis, we also examined the loss of mitochondrial membrane potential using JC-1. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations were performed on LoVo cells to examine the morphological changes and the ultrastructure modifications, respectively, after the treatment. Results and discussion Spermine and ZmPAO treatment reduced the number of colonies in a spermine dose-dependent manner and a time-dependent manner in LoVo and AGS cells. I also found that the treatment further decreased the cell viability in LoVo MDR cells when compared with LoVo WT cells. Moreover, there was no difference in cell viability after the treatment with spermine and spermidine in both LoVo WT and MDR cells, suggesting that aminoaldehydes derived from spermine and spermidine do not have any cytotoxic effect, it is mainly due to H2O2. Further colony formation assay revealed that cytotoxicity induced by spermine metabolites was completely prevented by the addition of exogenous catalase in both LoVo WT and MDR cells, while it was not affected by the presence of exogenous ALDH. Moreover, LoVo cells exposed to exogenous H2O2 exhibited a slightly higher cytotoxicity than that induced by ZmPAO and spermine. The concentration of H2O2 actually generated by ZmPAO from spermine might be lower than that of H2O2 when directly exposed. Similar results were also obtained in AGS cells, showing that the effect of cell growth inhibition induced by polyamines and ZmPAO was not cell specific. Treatment with ZmPAO and spermine at 42°C showed an increase in cytotoxicity on LoVo and AGS cells when compared with the cells treated at 37°C. Cytotoxicity was higher in LoVo MDR cells than in their WT counterpart, and it is probably due to a synergistic effect of spermine metabolites at 42°C. Annexin V-FITC and PI staining assay showed that the treatment with ZmPAO and spermine increased the percentage of apoptotic cells population in a spermine dose-dependent manner in LoVo WT. The treatment also increased the cell population at the sub-G1 phase that is characteristic of apoptosis. In both assays, the proportion of apoptotic cells was higher in LoVo MDR than in LoVo WT. SEM analysis showed a significant number of membrane blebs after the treatment with ZmPAO and spermine in LoVo MDR cells, while LoVo WT cells showed minor morphological changes. TEM observation further revealed that the treatment exhibited the typical ultrastructural features of apoptosis, including vacuolization in the cytoplasm and mitochondrial alterations. In particular, in MDR cells, I found severe vacuolization and cell membrane blebbing. The involvement of mitochondria in the cytotoxicity was supported by JC-1 assay. It showed that the exposure of LoVo cells to ZmPAO and spermine reduced mitochondrial membrane potential, which was greater in LoVo MDR cells than in WT cells, in agreement with the results of cell viability and apoptosis assays. We demonstrated that ZmPAO and spermine treatment induced cytotoxicity through an apoptotic pathway, which was more pronounced in LoVo MDR cells compared to LoVo WT ones. Since polyamine levels are up-regulated in tumor sites and the development of MDR tumor cells is recognized as a major problem, the design of a new therapeutic strategy based on the use of this combination in human tumors might be taken into account, making this approach mainly attractive in treating MDR cancer patients.