Synergistic activation of macrophage activity by polyamine deprivation and cyclophosphamide.

A synergistic reduction in tumor growth rate and of metastasis dissemination together with an increased life span, were observed when polyamine deprivation was combined with a low dose of cyclophosphamide. When rats were treated with this combination treatment, TNF and NO release was enhanced and phagocytic activity was increased. In addition, PGE2 release by macrophages was enhanced, whereas PGE2 plasma levels were restored to normal values. The decrease in PGE2 plasma levels presumably indicates the synergistic effect of the combination treatment on tumor cell metabolism. The enhancement of macrophage tumoricidal activities indicates immunostimulating synergistic effects, which may contribute to the anti-tumoral effect of the treatment.

Flow cytometric analysis of in vivo polyamine deprivation in Lewis lung carcinoma (3LL) cells using the monoclonal antibody SPM8-2.

It has previously been shown that the monoclonal antibody SPM8-2 recognizes free spermine and spermidine as well as polyamines bound by an amide bond. In the present work it is demonstrated that this antibody also interacts with spermidine, spermine, and to a lesser extent N1- and N8-acetyl spermidine in an ELISA test where the polyamines are bound by reaction with formaldehyde. 3LL Lewis lung carcinoma cells from tumor-grafted mice were labeled with fluorescein-conjugated monoclonal antibody SPM8-2 and analyzed by flow cytometry. Both viable cells and formaldehyde-fixed and subsequently permeabilized cells showed fluorescent staining. However, most polyamines present in the cells are not directly available for antibody binding. Treatment of fixed cells with DNase or RNase greatly increased fluorescent staining, suggesting that some polyamines are co-localized with DNA and RNA. Antibody labeling of the cells was prevented by addition of free spermine. 3LL cells from tumors of mice treated by a polyamine depleting regimen had decreased intracellular spermidine levels and bound less antibody when compared to untreated controls. After digestion with RNase, the cells from treated mice bound considerably less fluorescent antibody than tumor cells from untreated mice, while their RNA content was similar. In contrast, fluorescent staining after DNase digestion was only slightly affected by the treatment with a polyamine depleting regimen. This suggests that the pools of polyamines which are co-localized with RNA are depleted more readily than those associated with DNA. Since only a small proportion of the intracellular polyamines is accessible to the bulky antibodies, treatment with hydrolytic enzymes (DNase, RNase) is necessary to reveal specific compartments of the polyamines and to demonstrate qualitative and semi-quantitative differences of their distribution within cells.

Polyamine deprivation prevents the development of tumour-induced immune suppression.

Mice grafted with the 3LL (Lewis lung) carcinoma exhibit immune suppression: spleen cells showed decreased spontaneous interleukin 2 (IL-2) production and T-CD4+ and T-CD8+ lymphocyte populations; in addition the polyamine content in the spleen was increased. By treating the mice with a polyamine-deficient diet containing neomycin, metronidazole and inhibitors of ornithine decarboxylase and polyamine oxydase, tumour growth was reduced and the immune abnormalities were reversed. The spleen cells overproduced IL-2 by reducing exogenous sources of polyamines, but total blockade of all major polyamine sources was necessary to obtain an optimal effect both on IL-2 production and on spleen polyamine content. Irrespective of whether polyamine deprivation was started at an early or at an advanced stage of tumour growth, T-lymphocyte populations were restored to normal values, demonstrating that polyamine deprivation not only prevents tumour-induced immune suppression, but reverses established immunological disorders. In contrast to what was observed regarding IL-2 production by spleen cells and natural killer (NK) cell activity, the polyamine oxidase (PAO) inhibitor did not enhance the number of T lymphocytes. These findings are consistent with a direct effect of the polyamines on immune effector cell metabolism. They suggest an important role of the gastrointestinal polyamines and of PAO activity in the regulation of IL-2 production.

Observations with an S-adenosylmethionine decarboxylase inhibitor in rats with prostatic adenocarcinoma.

CGP 48664A (2-(4-aminoiminomethyl)-2,3-dihydro-1H-inden-1-ylidene dihydrochloride, CAS 149400-88-4) is a new potent inhibitor of S-adenosylmethionine decarboxylase with antitumor properties. In view of the eminent clinical problems in the treatment of non hormone dependent prostatic cancer, the antiproliferative potency of this compound was tested in Dunning MAT-LyLu rat prostatic adenocarcinoma. The compound proved inefficient in preventing the growth of this tumor, even at a near toxic dose. A reason for the lacking effect is presumably the inadequate accumulation of the drug in the tumor cells due to the excessive growth rate of the MAT-LyLu xenografts. Tumor growth seems not to be accompanied by a proportionally rapid vascularization of the tumor mass. CGP 48664A was found to be a potent inhibitor of polyamine oxidase. This property of the drug may have contributed to the activation of the phagocytic capacity of peritoneal macrophages from tumor-bearing rats.

In vivo, synergestic inhibition of MAT-LyLu rat prostatic adenocarcinoma growth by polyamine deprivation and low-dose cyclophosphamide.

Polyamine deprivation in vivo produces significant tumor growth inhibition of the hormone-resistant, metastatic Dunning Mat-LyLu murine prostatic carcinoma. In order to produce a cytotoxic effect in addition to the cytostatic effect of polyamine deprivation, various chemotherapy regimens, combined with drug-containing polyamine-deficient chow (DC-PDC), were assessed. Triple chemotherapy combining methotrexate, cyclophosphamide and vindesine; and monochemotherapy with high-dose cyclophosphamide (90 mg. kg-1) and low-dose cyclophosphamide (20 mg.kg-1) were studied alone and in combination with DC-PDC. A variant of DC-PDC excluding the polyamine oxidase inhibitor MDL 72527 was also studied in combination with low-dose cyclophosphamide. The triple-chemotherapy regimen alone or in combination with polyamine deprivation was effective on tumor growth inhibition but was also toxic. High-dose cyclophosphamide alone produced significant tumor growth inhibition and an increase in life span. High-dose cyclophosphamide in combination with DC-PDC was also effective on tumor growth but was also toxic. Low-dose cyclophosphamide alone was moderately effective on tumor growth inhibition with a marginal increase in life span. When combined with polyamine deprivation, results with low-dose cyclophosphamide compared favourably with those of high-dose cyclophosphamide alone and prevented the formation of lung metastases. The polyamine oxidase inhibitor does not appear to be mandatory to achieve this effect if DC-PDC is combined with low-dose cyclophosphamide. Polyamine deprivation appears to be an important tool in anticancer therapy, allowing the use of reduced doses of cytotoxic agents with the same antitumoral efficacy.

[The involvement of polyamines in the malignant proliferative process. The anticancer effect of polyamine deprivation]. / Implication des polyamines dans les processus prolifératifs malins. Effets anticancéreux d'une carence en polyamines.

The fact that tumors require polyamines for growth has been repeatedly demonstrated. In vivo polyamines are available both from endogenous (intracellular biosynthesis) and exogenous sources (food and intestinal microflora). We investigated in rats grafted with Mat-Lylu prostatic adenocarcinoma the distribution between tumor and tissues of orally administered (14C) putrescine (Pt). The amount of radioactivity retained by tumors was directly proportional to the tumor volume. In a tumor of 25 cm3 19% of the totally retained radioactivity was found. The accumulation of Pt by intestinal brush-border membrane vesicles prepared from tumor-bearing animals was significantly higher than by vesicles from healthy rats. Our results indicate that the presence of a tumor induces an adaptive response in the small intestine which stimulates the uptake of exogenous polyamines. Our therapeutic strategy was to realise a total blockade of all endogenous and exogenous sources of polyamines by feeding animals with a drug (DFMO, MDL 72527, antibiotics) containing polyamine deficient chow. We observed that polyamine deprivation largely reduced both primary tumor and metastatic development. Natural Killer cell cytotoxic activity and blood formula were restored to normal values after treatment. Furthermore polyamine deprivation enhanced anti-tumoral efficacy of chemotherapy.

[Involvement of polyamines in malignant proliferative processes: antineoplastic effects of a polyamine deficiency]. / Implication des polyamines dans les processus prolifératifs malins: effets anticancéreux d'une carence en polyamines.

The fact that tumors require polyamines for growth has been repeatedly demonstrated. In vivo polyamines are available both from endogenous (intracellular biosynthesis) and exogenous sources (food and intestinal microflora). We investigated in rats grafted with Mat-Lylu prostatic adenocarcinoma the distribution between tumor and tissues of orally administered [14C]putrescine (Pt). The amount of radioactivity retained by tumors was directly proportional to the tumor volume. In a tumor of 25 cm3 19% of the totally retained radioactivity was found. The accumulation of Pt by intestinal brush-border membrane vesicles prepared from tumor-bearing animals was significantly higher than by vesicles from healthy rats. Our results indicate that the presence of a tumor induces an adaptive response in the small intestine which stimulates the uptake of exogenous polyamines. Our therapeutic strategy was to realise a total blockade of all endogenous and exogenous sources of polyamines by feeding animals with a drug (DFMO, MDL 72527, antibiotics) containing polyamine deficient chow. We observed that polyamine deprivation largely reduced both primary tumor and metastatic development. Natural Killer cell cytotoxic activity and blood formula were restored to normal values after treatment. Furthermore polyamine deprivation enhanced antitumoral efficacy of chemotherapy.

Polyamine deprivation: a new tool in cancer treatment.

The fact that tumors require polyamines for growth has been demonstrated in vitro and in vivo and widely reported. This finding led to the use of polyamine biosynthetic enzymes as targets for antitumor drug design. Highly efficient in vitro selective inhibitors of ornithine decarboxylase such as DFMO do not produce important antitumoral effects in vivo, due to the ability of tumor cells to uptake extracellular polyamines. A new strategy was developed, combining a systematic blockade of all endogenous and exogenous sources of polyamines in vivo. Sources of exogenous polyamines were eliminated by administration of a polyamine-free diet to the animals and decontamination of their gastrointestinal tract. Important antitumoral effects were obtained with this polyamine deprivation and are presented with two experimental models of tumors (Lewis lung carcinoma, Mat Lylu prostatic carcinoma). Biological parameters, modified in cases of cancer, were restored to normal values in treated animals: blood counts and NK cytotoxic activity. Number of metastases was significantly reduced. Given that in man cancer treatment remains unsatisfactory due to incomplete cell kill, development of resistance to treatment and secondary effects of chemotherapy, we chose to investigate the potential interest of polyamine deprivation in this field. By combining clinically applied cytotoxic drugs with polyamine deprivation, we observed an improvement of their antitumoral efficiency: a considerable retardation of tumor growth paired with a marked increase in life-span of the treated animals. Our observations confirm that polyamines absorbed from exogenous sources, mainly food and gastrointestinal tract, play an important role in tumor growth control. Furthermore, the study shows that polyamine deprivation represents an important potential therapeutic tool in improved management of cancer treatment.

Polyamine deprivation stimulates natural killer cell activity in cancerous mice.

It has recently been established that the total blockade of all endogenous and exogenous sources of polyamines by a drug containing polyamine deficient chow (DC-PDC+) could inhibit tumor growth in vivo and increase the antitumoral efficacy of chemotherapy drugs. We show here that polyamine deprivation obtained with DC-PDC+ not only influences tumor development via reduction of polyamine concentrations in the tumor itself but, in addition, stimulates cells of the non-specific immune system specialized in tumor cell killing. We report that mice grafted with the 3LL carcinoma present a dramatic decrease in the cytotoxic activity of their natural killer (NK) cells. When these animals are treated with DC-PDC+, their NK cell activity is completely restored to normal values. Normalization of leucocyte number and differential count was observed as well. With respect to the different components of the DC-PDC+, it was observed that the endogenous and exogenous sources of polyamines have a different degree of impact on tumor development. For example, when only polyamines from digestive sources are deprived, a weak but significant improvement in NK activity and antitumoral effects were observed, without affecting intra-tumoral polyamine concentrations. We conclude that polyamines, secreted by the tumor itself as well as absorbed through the gastrointestinal tract, could now be considered not only as autocrine growth factors but also as natural immunosuppressive factors.

Hamster cells, untreated and treated with chemical carcinogens, maintained in vitro for 2 1/2 years.

We have maintained in culture, for a prolonged period, untreated hamster cells from whole embryo, foetal brain and lung from newborn animals. Among the 7 lines studied we observed only one spontaneous transformation during the first year of culture. The cells of the 6 other control lines remained normal and diploid, and were not transplantable during the first 9 to 12 months of culture. After the 12th month, changes appeared in their in vitro behaviour and their transplantability: grafts of 0-5-2 X 10(6) cells induced tumours in the hamster; fewer cells did not. In vitro chemically transformed hamster cells were fundamentally different from untreated cells of the same origin, not only in morphological and growth characteristics but also in transplantability; of the 9 lines obtained, 7 induced tumours after injection of 10(1)-10(4) cells, and 2 after injection of 10(5) cells per animal.