Elevated neutrophil elastase and acrolein-protein adducts are associated with W256 regression.

The involvement of granulocytes in immune response against cancer is not well understood. Depending on the cytokine milieu in which they act and on their oxidative burst, granulocytes may play either an inhibitory or stimulatory role in tumour growth. Unsaturated fatty acids, essential components of cellular membranes and storage lipids, are susceptible to granulocyte-derived reactive oxygen species (ROS). ROS can induce lipid peroxidation (LPO) resulting in the destruction of biomembranes. Thus, murine W256 tumour progressing and tumour regressing animal models were used to study the involvement of plasma inflammatory mediators and oxidative burst of circulating granulocytes in malignant destruction and detrimental tumour growth. The involvement of LPO-derived aldehydes (i.e. acrolein, 4-hydroxy-2-nonenal and malondialdehyde) and myeloperoxidase (MPO) appearance in the granulocyte anti-cancer response were further evaluated. The results obtained revealed a significant increase in neutrophil elastase in animals with regressing tumour. Furthermore, the presence of MPO in tumour microenvironment was accompanied by the formation of acrolein only 5 h after tumour transplantation and its presence increased during tumour regression. Later, at an early stage of tumour regression, the presence of other LPO-derived aldehydes were also observed. The results obtained suggest that elevated neutrophil elastase and initiation of LPO may play an important role in the tumour development leading to tumour regression.

Immunostimulatory effect of natural clinoptilolite as a possible mechanism of its antimetastatic ability.

PURPOSE: Many biochemical processes are closely related to ion exchange, adsorption, and catalysis. Zeolites reversibly bind small molecules such as oxygen or nitric oxide; they possess size and shape selectivity, the possibility of metalloenzyme mimicry, and immunomodulatory activity. These properties make them interesting for pharmaceutical industry and medicine. METHODS: The experiments were performed on mice. Different biochemical and molecular methods were used. RESULTS: Micronized zeolite (MZ) administered by gastric intubation to mice injected with melanoma cells significantly reduced the number of melanoma metastases. In mice fed MZ for 28 days, concentration of lipid-bound sialic acid (LSA) in serum increased, but lipid peroxidation in liver decreased. The lymphocytes from lymph nodes of these mice provoked a significantly higher alogeneic graft-versus-host (GVH) reaction than cells of control mice. After i.p. application of MZ, the number of peritoneal macrophages, as well as their production of superoxide anion, increased. However, NO generation was totally abolished. At the same time, translocation of p65 (NFkappaB subunit) to the nucleus of splenic cells was observed. CONCLUSION: Here we report antimetastatic and immunostimulatory effect of MZ and we propose a possible mechanism of its action.

Natural zeolite clinoptilolite: new adjuvant in anticancer therapy.

Natural silicate materials, including zeolite clinoptilolite, have been shown to exhibit diverse biological activities and have been used successfully as a vaccine adjuvant and for the treatment of diarrhea. We report a novel use of finely ground clinoptilolite as a potential adjuvant in anticancer therapy. Clinoptilolite treatment of mice and dogs suffering from a variety of tumor types led to improvement in the overall health status, prolongation of life-span, and decrease in tumors size. Local application of clinoptilolite to skin cancers of dogs effectively reduced tumor formation and growth. In addition, toxicology studies on mice and rats demonstrated that the treatment does not have negative effects. In vitro tissue culture studies showed that finely ground clinoptilolite inhibits protein kinase B (c-Akt), induces expression of p21WAF1/CIP1 and p27KIP1 tumor suppressor proteins, and blocks cell growth in several cancer cell lines. These data indicate that clinoptilolite treatment might affect cancer growth by attenuating survival signals and inducing tumor suppressor genes in treated cells.

Involvement of lipid peroxidation, oncogene expression and induction of apoptosis in the antitumorous activity of ferric-sorbitol-citrate.

We described before that iron-containing, anti-anaemic drug, ferric-sorbitol-citrate complex (FSC) inhibited proliferation of various murine cancer cells in vitro and caused tumour regression in vivo, but did not affect proliferation of the non-malignant cells. The aim of this study was to evaluate further the anticancer activity mechanism of FSC using human colon cancer cell line CaCo2. After treatment with FSC for 72 hours impaired proliferative ability and viability of CaCo2 cells as observed. Growth modification caused by FSC involved diminished expression of Bcl-2, and over-expression of mp53 proto-oncogenes, accompanied by increased incidence of apoptosis. Immunostaining the cells applying monoclonal antibodies for lipid peroxidation product 4-hydroxynonenal (HNE) showed that FSC-iron increased intracellular HNE, but did not induce severe HNE-mediated oxidative stress. Thus, antitumorous mechanism of FSC involves modulation of oncogene expression and induction of apoptosis apparently not triggered by lipid peroxidation-mediated oxidative stress, although FSC might restore endogenous HNE production in the CaCo2 cells to level resembling physiological for various non-malignant cells and tissues. Higher dose of FSC increased also number of intracellular ferritin positive CaCo2 cells.

Ferrocenes (F168, F169) and fero-sorbitol-citrate (FSC): potential anticancer drugs.

We have shown earlier that the iron containing, ferric-sorbitol-citrate complex (FSC) inhibited proliferation of cultured mouse melanoma B16, GHC, KB, HeLa and CaCo2 cells and caused mouse melanoma regression in vivo. This drug did not affect the proliferation of the nonmalignant fibroblast L929 line, human bone marrow-HBS, VERO and HEF cell line. It is also known, that some metallocene derivatives posses antitumour properties resulting principally from their action on the metabolism of DNA, and subsequently, RNA and proteins. We synthesized in our laboratory some ferrocene analogs (F168 and F169) and tested their antiproliferative ability for malignant human carcinoma Hep2 and mouse melanoma F10 cell lines. As control cell lines, human HEF and mice L929 fibroblasts were used. The tested iron substances were very potent in inhibiting the growth of malignant cell lines, whereas they had no significant inhibitory effect on the viability of nonmalignant fibroblasts. The most pronounced growth inhibitory and cytotoxic effect was found in the malignant F10 cells and the most potent was ferrocene F169. Because of their selective effect on malignant cells, the ferric-sorbitol-citrate complex as well as tested ferrocenes will be further investigated and submitted as new antitumour substances.

[The role of iron in neoplasms]. / Uloga zeljeza u neoplazmi.

The purpose of this review is to concisely summarize the current knowledge of iron participation in the carcinogenic process, especially from the standpoint of redox regulation, and to introduce a hypothesis for the mechanism. In both animals and humans, primary neoplasms develop more frequently at body sites of excessive iron deposits. Iron exerts its carcinogenic effects by catalysing formation of hydroxil radicals, suppressing activity of host defence cells and promoting cancer cell multiplication. Manipulations of cellular iron metabolism for modulating normal and malignant cell proliferation is described. Quantitative evaluation of body iron and iron-withholding proteins has prognostic value in cancer patients. Procedures associated with lowering host iron intake and inducing host cell iron efflux can assist in prevention and management of neoplastic disease.

Impaired proliferation and DNA synthesis of a human tumor cell line (HeLa) caused by short treatment with the anti-anemic drug jectofer (ferric-sorbitol-citrate) and the lipid peroxidation product 4-hydroxynonenal.

Anti-anaemic drug, ferric-sorbitol-citrate complex (FSC), inhibit tumour cell growth through the mechanisms which are complex and not entirely understood. The probable mechanisms of described effects of iron is iron-induced oxidative stress of the treated cells. Hence, the effects of FSC on HeLa cell growth in vitro were compared with the biological activity of one of the major mediators of the oxygen free radicals--aldehyde 4-hydroxinonenal (HNE), to see if the effects of FSC and of HNE resemble each other. Impaired proliferative ability and DNA synthesis of HeLa cells was observed after treatment with anti-anaemic drug FSC for 24 hours. After treatment with FSC and culturing of HeLa cells in fresh medium for 24 or 96 hours the cells did not proliferate at all, DNA synthesis was transiently recovered and then diminished again. HNE blocked cell proliferation during the time the aldehyde was present in culture and 24 h later. Afterwards, the cells proliferated as control non-treated cells. HNE did not inhibit DNA synthesis during treatment, but intensity of 3H-thymidine incorporation was lower after preincubation. Thus, both FSC and HNE interfere with the basic mechanisms of the cell growth regulation, while antitumour activity of FSC resembles, but does not necessarily include iron induced lipid peroxidation.

Presence of c-Myc protein in murine myeloid leukaemia cells during growth and after irradiation.

Myeloid leukaemia (ML) is strain specific for RFM mice, which were used in these experiments. We investigated the presence of c-myc protein during the growth of ML and after irradiation of leukaemic cells. Leukaemic spleen cells were investigated 9 (nonterminal phase NTP) or 12 days (terminal phase TP) after the injection of ML cells. Leukaemic cells of NTP were irradiated with X-rays or UV-light. c-Myc protein was detected by immunocytochemical method. c-Myc protein was expressed in 74.98% of spleen cells of healthy RFM mice. In the early period of leukaemia growth (NTP) only 14.33% of c-myc positive cells were found, as opposed to the terminal phase (TP) of leukaemia when 89.7% of c-myc positive cells were detected. These results indicated the connection of growth of ML and the presence of c-myc protein. If the spleen cells of NTP of leukaemia were irradiated by X-rays or UV-light, the number of cells which expressed c-myc protein was extremely increased.

The mechanism of recurrence of mouse myeloid leukaemia after total body irradiation and bone marrow transplantation.

RFM mice were X-irradiated (9.5 Gy) 3, 4, 5, 6 or 7 days after inoculation of a transplantable strain-specific myeloid leukaemia (ML) and were reconstituted or not with syngeneic or allogeneic bone marrow cells. Recurrent leukaemia was observed in mice with either type of the bone marrow transplant, indicating that ML cells survived the dose of 9.5 Gy of X-rays. ML cells exposed in vitro to high doses of X-rays (20, 30, 40, 50, 60 Gy) and injected into lethally irradiated syngeneic recipients were still capable of forming leukaemic colonies on the spleens. Higher doses (70, 80, 90 and 100 Gy) abolished the colony formation completely. Irradiated ML cells were also capable of causing leukaemia (hepatosplenomegaly) if inoculated into lethally irradiated CBA mice reconstituted with bone marrow cells of CBA or RFM mice. That was attributed to the release of a leukaemogenic factor from the ML cells, capable of transforming transplanted normal cells.

Inhibition of IL-1 activity induced with allogeneic transfusion of UV-irradiated blood.

Treatment with UV-irradiated donor-specific blood transfusion is known to induce specific unresponsiveness in recipient animals and prolong allograft survival. Mixed lymphocyte response in transfused mice was decreased towards spleen cells of the blood donor strain, but was not altered to third-party cells. Sera from treated mice showed significantly lower interleukin-1 (IL-1) activity, which was increased with higher dilutions of sera, indicating the presence of IL-1 inhibitor. Furthermore, sera decreased rIL-1-induced cell proliferation in dose-dependent manner, while the response to rIL-2 neither depended on the concentration of sera, nor differed between non-treated controls and treated mice. These results indicate that UV-irradiated allogeneic blood transfusion could induce an inhibitor, specifically directed to IL-1 activity, which may be involved in the generation of immunological unresponsiveness in treated animals.

Influence of iron on proliferation and cell cycle kinetics on cultured malignant and nonmalignant cells.

The presence of an iron-containing complex (FSC, ferric sorbitol citrate) in medium inhibited proliferation of malignant (KB. GHC) cells: but did not appreciably alter the proliferation of normal (HBS, Vero) cells. Flow cytometric analysis showed considerable differences of malignant and normal cell growth kinetics. With addition of 200 microM Fe in FSC, malignant cell proliferation was suppressed. An increased number of cells in G1 and early S phase suggested that iron excess blocked the cell cycle before beginning of DNA synthesis.

Immune response of stressed rats treated with drugs affecting serotoninergic and adrenergic transmission.

Stressful conditions interfere with immune response. One of the principal mechanisms is activation of hypothalamo-pituitary-suprarenal axis by central serotoninergic and adrenergic pathways. Alternative mechanisms bypassing the axis also take part in stress-induced immunomodulation. Immunosuppression caused by repeated restraints or over-crowding was usually accompanied by increased metabolism of serotonin in the brain (as indicated by increased level of its metabolite, 5-HIAA) and by increased levels of corticosterone in plasma. Changes in lymphatic tissues of stressed animals that result in suppression of immune response apparently "outlive" fluctuating changes in neurotransmitter and corticosterone levels. Drugs that alter serotoninergic or adrenergic transmission interfere with immunosuppressive effect of stress either synergistically (augmenting suppression) or antagonistically (preventing it). Since immunocompetent cells possess serotoninergic and adrenergic receptors, such drugs may exert their effect either via central neuroendocrine mechanisms, or by direct effects on immunocompetent cells.

Resistance of human and mouse myeloid leukemia cells to UV radiation.

Sensitivity of mouse bone marrow and myeloid leukemia cells as well as the sensitivity of human myeloid leukemia cells to UV light was tested. Criteria were the in vivo colony-forming ability of UV exposed cells and the inhibition of DNA synthesis during post-irradiation incubation for 24 h in vitro. Mouse bone marrow cells irradiated with a small dose of UV light (5 J/m2) and injected into x-irradiated animals did not form hemopoietic colonies on the recipients' spleens, and the recipients died. However, mouse leukemia cells, after irradiation with higher doses of UV light, retained the ability to form colonies on the spleens, and all recipient mice died with typical symptoms of leukemia. In vitro, mouse bone marrow cells exhibited high sensitivity to UV light as compared to mouse myeloid leukemia cells. Human leukemia cells were also resistant to UV light, but more sensitive than mouse leukemia cells. These results indicate that myeloid leukemia cells are resistant to UV light as compared with normal bone marrow cells.

Influence of the thymus extract on the immunological function of animals with experimental diabetes.

After immunization with SRBC, the number of plaque-forming cells (PFC) in the spleen of alloxan-diabetic mice, in nondiabetic TIR mice and in alloxan-diabetic TIR mice was significantly decreased as compared with control non-diabetic donors. The ability of lymphocytes from alloxan-diabetic mice to adoptively restore the suppressed immune response of TIR mice, was reduced in comparison with the effect of lymphocytes from normal, nondiabetic donors. Local GVH reaction in nondiabetic rat recipients provoked by lymphocytes from control healthy mice was 5.6 +/- 0.7 mm. Significantly lower rate of local GVH reaction after injection of lymphocytes from diabetic donors was found in diabetic as in nondiabetic recipients as well. Treatment of alloxan-diabetic mice with thymus extract or with insulin, partly restored depressed function of the humoral and cellular system. Treatment of diabetic mice with both thymus extract and insulin, was even more effective in restoring of their immune reactivity. Diabetic condition strongly influenced the function of the immune system. This could be attributed to depletion of T-lymphocytes, changed relations between the lymphocyte subpopulations in diabetic donors, and disturbance of lymphocyte metabolism.

Suppression of immune response in rats by stress and drugs interfering with metabolism of serotonin.

Rats immunized with sheep erythrocytes were stressed by repeated restraint and/or treated with a precursor of serotonin (5-hydroxytryptophan, 5-HTP) or with an inhibitor of serotonin synthesis (parachlorophenylalanine, PCPA). As expected, repeated stress reduced the plaque-forming cell (PFC) response. Treatment with 5-HTP also reduced the PFC response, and potentiated the immunosuppressive effect of stress. This was accompanied by increased metabolism of serotonin in the brain, as indicated by increased concentration of its metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in cerebral tissue. Treatment with PCPA also suppressed the PFC response, but this suppression was accompanied by decreased levels of brain serotonin and of 5-HIAA. Plasma corticosterone levels were elevated, reaching statistical significance, in rats treated with PCPA. Both drugs suppressed the in vitro PFC response of peripheral blood lymphocytes. Thus, although 5-HTP and PCPA altered serotonin metabolism in the brain in a diametrically opposite manner, their effects on the immune response were the same. Putative central effects of the drugs on serotoninergic regulation of the immune response were apparently obscured by their effects on corticosterone secretion as well as by their direct effects on immunocompetent cells.

Effect of diazepam on brain neurotransmitters, plasma corticosterone, and the immune system of stressed rats.

Rats were treated with injections of diazepam (1 or 10 mg/kg) and stressed by restraint lasting 3 hours. This was performed once or, in animals immunized with sheep erythrocytes, repeatedly for 4 consecutive days. After repeated stress and/or diazepam treatment, the levels of brain noradrenalin decreased in all treated groups. Although both treatments (stress and diazepam) diminished the 5-hydroxytryptamine (5-HT)/5-hydroxyindoleacetic acid (5-HIAA) ratio, treatment with either dose of diazepam prevented the stress-induced fall of this ratio. The activity of hypothalamic glutamate decarboxylase, the enzyme taking part in GABA synthesis, was affected neither by the acute nor by repeated stress and/or diazepam treatment. The levels of plasma corticosterone were enhanced in all stressed rats, with and without drug. This finding was in accordance with the enhanced weights of adrenal glands in repeatedly stressed rats. The tendency to a corticosterone rise after repeated treatment with diazepam, 10 mg/kg, coincided with the enhanced weights of adrenal glands in these animals. The plaque-forming cell (PFC) response was reduced in all stressed animals and in animals treated with diazepam, 10 mg/kg. Accordingly, high doses of diazepam given repeatedly to rats are immunosuppressive, achieving this effect presumably by an enhancement of glucocorticoid secretion. Neither the low nor the high doses of diazepam affect the stress-induced enhancement of hypothalamohypophysial-adrenal axis activity and consecutive immunosuppression.

Suppression of the immune response by drugs interfering with the metabolism of serotonin.

The work was based on the assumption that neurohumoral control of the immune response, particularly in stressed animals, involves central serotoninergic mechanisms. Rats immunized with sheep erythrocytes were stressed by repeated restraints and/or treated with a precursor of serotonin (5-hydroxytryptophan, 5-HTP) or with an inhibitor of serotonin synthesis (parachlorophenylalanine, PCPA). As expected, repeated stresses reduced the plaque-forming cell (PFC) response. Treatment with 5-HTP also reduced the PFC response, and potentiated the immunosuppressive effect of stress. This was accompanied by increased metabolism of serotonin in the brain, as indicated by increased concentration of its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in cerebral tissue. Treatment with PCPA also suppressed the PFC response, but this suppression was accompanied by decreased levels of brain serotonin and of 5-HIAA. Plasma corticosterone levels were elevated in rats treated with PCPA. It seems that putative central effects of PCPA on serotoninergic regulation of the immune response were outweighed by its effects on corticosterone secretion and/or on lymphoid cells.