Nutritional shortage augments cisplatin-effects on murine melanoma cells.

Melanoma incidence increases every year worldwide and is responsible for 80% of skin cancer deaths. Due to its metastatic potential and resistance to almost any treatments such as chemo, radio, immune and targeted-therapy, the patients still have a poor prognosis, especially at metastatic stage. Considering that, it is crucial to find new therapeutic approaches to overcome melanoma resistance. Here we investigated the effect of cisplatin (CDDP), one of the chemotherapeutic agents used for melanoma treatment, in association with nutritional deprivation in murine melanoma cell lines. Cell death and autophagy were evaluated after the treatment with cisplatin, nutritional deprivation and its association using an in vitro model of murine melanocytes malignant transformation to metastatic melanoma. Our results showed that nutritional deprivation augmented cell death induced by cisplatin in melanoma cells, especially at the metastatic subtype, with slight effects on melanocytes. Mechanistic studies revealed that although autophagy was present at high levels in basal conditions in melanoma cells, was not essential for cell death process that involved mitochondrial damage, reactive oxygen species production and possible glycolysis inhibition. In conclusion, nutritional shortage in combination with chemotherapeutic drugs as cisplatin can be a valuable new therapeutic strategy to overcome melanoma resistance.

Apoptosis induced by Aß25-35 peptide is Ca(2+) -IP3 signaling-dependent in murine astrocytes.

Although the accumulation of the neurotoxic peptide ß-amyloid (Aß) in the central nervous system is a hallmark of Alzheimer's disease, whether Aß acts in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that cytosolic Ca(2+) dysregulation, induced by a neurotoxic fragment (Aß25-35), caused apoptosis in a concentration-dependent manner, leading to cytoplasmic Ca(2+) mobilization from extra- and intracellular sources, mainly from the endoplasmic reticulum (ER) via IP3 receptor activation. This mechanism was related to Aß-mediated apoptosis by the intrinsic pathway because the expression of pro-apoptotic Bax was accompanied by its translocation in cells transfected with GFP-Bax. Aß-mediated apoptosis was reduced by BAPTA-AM, a fast Ca(2+) chelator, indicating that an increase in intracellular Ca(2+) was involved in cell death. Interestingly, the Bax translocation was dependent on Ca(2+) mobilization from IP3 receptors because pre-incubation with xestospongin C, a selective IP3 receptor inhibitor, abolished this response. Taken together, these results provide evidence that Aß dysregulation of Ca(2+) homeostasis induces ER depletion of Ca(2+) stores and leads to apoptosis; this mechanism plays a significant role in Aß apoptotic cell death and might be a new target for neurodegeneration treatments.

Interplay between apoptosis and autophagy, a challenging puzzle: new perspectives on antitumor chemotherapies.

Autophagy is a mechanism of protection against various forms of human diseases, such as cancer, in which autophagy seems to have an extremely complex role. In cancer, there is evidence that autophagy may be oncogenic in some contexts, whereas in others it clearly contributes to tumor suppression. In addition, studies have demonstrated the existence of a complex relationship between autophagy and cell death, determining whether a cell will live or die in response to anticancer therapies. Nevertheless, we still need to complete the autophagy-apoptosis puzzle in the tumor context to better address appropriate chemotherapy protocols with autophagy modulators. Generally, tumor cell resistance to anticancer induced-apoptosis can be overcome by autophagy inhibition. However, when an extensive autophagic stimulus is activated, autophagic cell death is observed. In this review, we discuss some details of autophagy and its relationship with tumor progression or suppression, as well as role of autophagy-apoptosis in cancer treatments.

The role of mitochondrial function in glutamate-dependent metabolism in neuronal cells.

Glutamate is an important neurotransmitter in neurons and glial cells and it is one of the keys to the neuron-glial interaction in the brain. Glutamate transmission is strongly dependent on calcium homeostasis and on mitochondrial function. In the present work we presented several aspects related to the role of mitochondria in glutamate signaling and in brain diseases. We focused on glutamateinduced calcium signaling and its relation to the organelle dysfunction with cell death processes. In addition, we have discussed how alterations in this pathway may lead or aggravate a variety of neurodegenerative diseases. We compiled information on how mitochondria can influence cell fate during glutamate stimulation and calcium signaling. These organelles play a pivotal role in neuron and glial exchange, in synaptic plasticity and several pathological conditions related to Aging, Alzheimer's, Parkinson's and Huntington's diseases. We have also presented autophagy as a mechanism activated during mitochondrial dysfunction which may function as a protective mechanism during injury. Furthermore, some new perspectives and approaches to treat these neurodegenerative diseases are offered and evaluated.

Bcl-2 expression and apoptosis induction in human HL60 leukaemic cells treated with a novel organotellurium(IV) compound RT-04.

Organotellurium(IV) compounds have been reported to have multiple biological activities including cysteine protease-inhibitory activity, mainly cathepsin B. As cathepsin B is a highly predictive indicator for prognosis and diagnosis of cancer, a possible antitumor potential for these new compounds is expected. In this work, it was investigated the effectiveness of organotellurium(IV) RT-04 to produce lethal effects in the human promyelocytic leukaemia cell line HL60. Using the MTT tetrazolium reduction test, and trypan blue exclusion assay, the IC50 for the compound after 24 h incubation was 6.8 and 0.35 microM, respectively. Moreover, the compound was found to trigger apoptosis in HL60 cells, inducing DNA fragmentation and caspase-3, -6, and -9 activations. The apoptsosis-induced by RT-04 is probably related to the diminished Bcl-2 expression, observed by RT-PCR, in HL60-treated cells. In vivo studies demonstrated that the RT-04 treatment (2.76 mg/kg given for three consecutive days) produces no significant toxic effects for bone marrow and spleen CFU-GM. However, higher doses (5.0 and 10 mg/kg) produced a dose-dependent reduction in the number of CFU-GM of RT-04-treated mice. These results suggest that RT-04 is able to induce apoptosis in HL60 cells by Bcl-2 expression down-modulation. Further studies are necessary to better clarify the effects of this compound on bone marrow normal cells.

Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom.

Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.

Cytotoxicity of materials used in perforation repair tested using the V79 fibroblast cell line and the granulocyte-macrophage progenitor cells.

AIM: To compare the cytotoxicity of materials used to repair perforations using permanent V79 fibroblasts and murine granulocyte-macrophage progenitor cells (CFU-GM). METHODOLOGY: Set specimens from amalgam, glass-ionomer, SuperEBA, N-Rickert, MTA and gutta-percha were eluted with culture medium for 72 h and their cytotoxicities were assessed by incubating the extracts with V79 and bone marrow-derived progenitors for 24 h and 7 days, respectively. Cytotoxicity on V79 cells was judged using the total nucleic acid content (NAC), neutral red uptake (NRU) and reduction of the tetrazolium salt (MTT). The number of bone marrow CFU-GM colonies determined in clonal cultures stimulated with recombinant murine granulocyte-macrophage colony-stimulating factor was used to assess cytotoxicity to progenitor cells. Statistical analyses were conducted using the one-way analysis of variance and Tukey's test where appropriate. RESULTS: All materials were cytotoxic in both cell systems; however, CFU-GM was more sensitive to the extracts than V79 cells. A similar rank order of toxicity was observed in V79 cells using the NAC and the MTT assays: glass-ionomer > N-Rickert congruent with SuperEBA > gutta-percha > amalgam congruent with MTA (P < 0.05). In contrast, the NRU test exhibited a lower sensitivity to MTA, gutta-percha and amalgam extracts. In the clonal culture assay, the toxicity was less pronounced in the presence of gutta-percha, SuperEBA and MTA. Similar cellular responses were found by placing the set specimens directly in the clonal culture dishes. CONCLUSIONS: The sensitivity of toxicity depended on the choice of the endpoint and the cell-culture system. Nevertheless, MTA was ranked as the least cytotoxic cement in both cell systems.

Ehrlich ascites tumor as a tool in the development of compounds with immunomodulatory properties.

In previous works, we have demonstrated that the myeloprotective properties of several natural and synthetic compounds are partly responsible for their antitumor activity in the Ehrlich ascites tumor (EAT) model. In this work, we present information that may be useful to the study of pharmacological and toxicological properties of compounds that affect the hematological compartment. Clonogenic studies in EAT-inoculated mice demonstrated a rapid decrease in bone marrow CFU-GM, whereas a progressive increase in splenic CFU-GM and cellularity was observed, followed by splenomegaly. Bone marrow cellularity declined on the third day after tumor challenge, returning to normal values thereafter. Serum from EAT-bearing mice produced detectable colony-stimulating activity in vitro. Similar results were observed with the conditioned medium from Ehrlich tumor cell cultures, but not with the cell-free Ehrlich tumor ascitic fluid. Tumor inoculation also resulted in a more striking depletion in the number of non-adherent cells in long-term bone marrow cell cultures (LTBMCs) with no bone marrow stroma formation. We speculate that the physiological alterations induced by the EAT growth can be used to assess the ability of compounds to modulate the hematopoietic response.

Hydroxyurea promotes the reduction of spontaneous BFU-e to normal levels in SS and S/beta thalassemic patients.

We have studied the effects of hydroxyurea on growth and differentiation of early erythroid progenitor cells (BFU-e) from peripheral blood of sickle cell disease patients (five SS and two Hb S/beta-thalassemia) in the presence or absence of exogenous stimulating factors. When the mononuclear cells from the sickle cell disease patients were cultured at diagnosis (before hydroxyurea treatment), there was an increased number of BFU-e in relation to controls (p < 0.05, Wilcoxon test) when cells were grown in the presence or absence of 5637 conditioned medium and erythropoietin. Colonies that developed in the absence of added growth factors were considered "spontaneous". A significant difference was observed after hydroxyurea treatment in the number of BFU-e obtained in the presence and absence of stimulus, with a higher reduction in the spontaneous BFU-e number. As expected, there was an increased Hb F level in these patients when compared with their pretreatment levels. There was no correlation between spontaneous BFU-e and hemoglobin levels in all patients studied.

Presence of micronuclei in lymphocytes of mercury exposed workers.

We have investigated the presence of micronuclei in mercury exposed workers. The study group consisted of 15 workers from a mercury-producing plant, mean age 39.5 years and a mean exposed period of 12 years. At the time of testing and for the six previous months, the exposed population had urinary mercury levels below the currently accepted limit of 50 ug/g creatinine. A significant increase in the percentage of micronuclei was observed in the mercury exposed individuals when compared to the non exposed group. We have not found any correlation between the percentage of micronuclei and age, length of exposure or urinary mercury concentrations. Our results suggest a genotoxic effect of mercury, which is observed in workers exposed chronically to levels considered biologically safe for the exposed population.

Haematopoietic response and bcl-2 expression in patients with acute myeloid leukaemia.

Bcl-2 expression, the number of apoptotic cells and the growth and differentiation of early bone marrow progenitor cells were studied in patients with confirmed diagnosis of acute myeloid leukaemia (AML). Bone marrow cells from normal individuals were used as controls. We observed an increased percentage of bcl-2-mononuclear bone marrow cells expression in AML patients in relation to controls (p =0.002). Accordingly, the number of apoptotic cells was reduced (p = 0.001) and there was a negative correlation between bcl-2 expression and the number of apoptotic cells (r=-0.664, p<0.001). In addition, bcl-2 expression was significantly increased in the chemotherapy resistant group in relation to the responsive group (p = 0.03). Lower rate of survival was observed in the group of AML patients with autonomous proliferation (p=0.01). These results suggest that a high bcl-2 expression and the presence of autonomous proliferation are related to a poor prognosis in AML.

Autonomous proliferation and bcl-2 expression involving haematopoietic cells in patients with myelodysplastic syndrome.

In this work, we investigated the autonomous proliferation, bcl-2 expression and number of apoptotic cells in the bone marrow of patients with confirmed diagnosis of myelodysplastic syndromes (MDS). Normal bone marrow cells obtained from donors of the Clinical Hospital of this university were used as a control. The autonomous proliferation, evaluated by clonal culture without exogenous growth factor, and the number of apoptotic cells in bone marrow kept for 10 days in liquid cultures at 37 degrees C and 5% carbon dioxide, were significantly greater in MDS patients than in control subjects (P = 0.001, Wilcoxon). However, bcl-2 expression, measured by immunocytochemistry, was significantly lower in MDS patients than in normal individuals (P = 0.002, Wilcoxon). These results suggest that the high proliferation activity in MDS patients may be counteracted by the high level of medullar cell death, which might be related to the lower bcl-2 expression.

Immunoglobulin levels in workers exposed to hexachlorobenzene.

The serum immunoglobulin (IgG, IgM and IgA) concentrations of 52 chlorinated-exposed workers were examined and compared with those of non-exposed, age- and sex-matched individuals. At the time of testing, the exposed population had mean hexachlorobenzene (HCB) blood levels of 3.84 micrograms/dl with a range of 0.1 to 16 micrograms/dl. Increased IgG and IgM levels were found in the HCB-exposed workers (P < 0.05 and P < 0.01, respectively). Hepatic function was evaluated by serum aspartate (AST) and alanine aminotransferase (ALT) activities, as well as by bilirubin levels. IgM concentrations were positively correlated with three of the studied parameters, namely, length of exposure (r = 0.367) and the activities of both AST (r = 0.367) and ALT (r = 0.507).

Defective neutrophil function in workers occupationally exposed to hexachlorobenzene.

In this work we have studied the respiratory burst and chemotaxis of polymorphonuclear leukocytes from 51 workers exposed to chlorinated compounds, which were compared with those of non-exposed, age- and sex-matched individuals. These two neutrophil functions were significantly reduced as compared to controls. No correlation was observed between the length of exposure, hexachlorobenzene (HCB) blood concentrations and neutrophil chemotaxis or the extent of nitroblue tetrazolium reduction.

The effect of lead on the bone marrow stem cells of mice infected with Listeria monocytogenes.

We investigated the effects of lead exposure on the growth and differentiation of bone marrow hematopoietic cells, the so called colony-forming cells, in normal and Listeria monocytogenes infected mice (resistant and susceptible strains). We also studied the effects of lead on the serum colony-stimulating activity (CSA), as well as on the survival of the mice after the infection. The doses of lead acetate were 13, 130 and 1300 ppm for 10, 30 and 70 d. At the end of this dosing, mice were infected with Listeria monocytogenes and killed 24, 48 or 72 h after inoculation of the bacteria. A dose-response suppressive effect of lead was observed in both strains in the 3 periods studied. However, in the resistant strain of mice the suppressive effects were overcome 48 h after the administration of the bacteria, whereas in the susceptible mice the suppressive effect of the infection was evident in all 3 time periods. The administration of lead caused no changes in serum hematopoietic growth factors, thus suggesting this metal acts by direct action on the myelopoietic cells. A significant decrease in host resistance, as measured by the mortality rate, was found when both strains of mice were challenged with sub-lethal doses of Listeria monocytogenes. Lethality was determined for a period of 10 d after dosing with 1300 ppm lead for 30 d.

Engulfment and killing capabilities of neutrophils and phagocytic splenic function in persons occupationally exposed to lead.

Phagocytosis and intracellular killing of Candida albicans and Candida pseudotropicalis by neutrophils as well as phagocytic splenic function from lead-exposed workers were studied. Two species of Candida were used since in individuals with myeloperoxidase deficiency neutrophils are unable to kill C. albicans, whereas C. pseudotropicalis can be effectively lysed. Phagocytosis with both antigens and phagocytic splenic function were normal in all the workers studied. However, lytic activity towards C. albicans, but not C. pseudotropicalis was impaired. This defect was observed in lead-exposed workers with blood lead levels and urinary delta-aminolevulinic acid (ALA-U) concentrations in the "safe" (below 60 micrograms/dl and 6 mg/l, respectively) and toxic ranges. An impaired ability to kill C. albicans suggests that lead exposure may lead to a myeloperoxidase deficiency. With the exception of blood lead levels and ALA-U concentrations, there was no correlation between any of the other parameters examined.

Immunoglobulin levels and cellular immune function in lead exposed workers.

The immunological status of lead acid battery workers with blood lead levels and urinary delta-aminolevulinic acid (ALA-U) concentrations ranging from safe to toxic levels has been examined and compared with those of non-exposed, age and sex matched controls. No differences in the serum concentrations of IgG, IgA and IgM between the populations were observed and there existed no correlation between blood lead level or ALA-U concentrations and serum immunoglobulin levels. In addition assessment was made of the capacity of peripheral blood mononuclear cells to respond to the mitogen phytohaemagglutinin (PHA), a correlate of T cell function. As before, there was no difference between exposed and control populations and no correlation between reactivity and blood lead concentration. Our data suggest that chronic exposure to lead fail to compromise lymphocyte function in man.