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Aims: Lactic acid bacteria (LAB) are known to have antitumoral properties, although the intrinsic mechanisms responsible for the execution of this activity are poorly understood. Here, we investigated the ability of concentrated supernatants from Lactobacillus plantarum to promote cell death in a human promyelocytic cell line., Methods and Results: We aim to test the hypothesis that concentrated supernatants from Lact. plantarum at 5, 50 or 100 μg/ml for 24 h exert cytotoxic effects on HL-60 cells. Accumulation of reactive oxygen species (ROS) was diminished and nuclear staining with Hoechst 33342 and propidium iodide (PI) determined a necrotic induction in a concentrationdependent sequence. Concentrated supernatants did not modify or reduced the activity of caspase-3. The assessment of phosphatidylserine externalization by annexin V/PI double staining led to a necrotic state, but the treatment did not produce a dissipation of mitochondrial membrane potential (ΔΨm), whereas cell cycle analysis revealed that concentrated supernatants failed to significantly enhance the population of HL-60 cells in the hypodiploid (sub-G1) fraction., Conclusions: Concentrated supernatants from Lact. plantarum are capable of inducing necrosis rather than apoptosis at high doses in a promyelocytic cell line., Significance and Impact of the Study: Here, we demonstrate the cytotoxic properties of concentrated supernatants from Lact. plantarum on a tumor cell line, and then, to open the possibility to analyze the chemical composition to elucidate the bioactive molecules.

Most antimicrobial peptides have been shown to have antitumoral activity. Cecropin A, a linear 37-residue antimicrobial polypeptide produced by the cecropia moth, has exhibited cytotoxicity in various human cancer cell lines and inhibitory effects on tumor growth. In this study, we investigated the apoptosis induced by cecropin A in the promyelocytic cell line HL-60. Treatment of cells with cecropin A was characterized by loss of viability in a dose-dependent manner, lactate dehydrogenase (LDH) leakage, and modest attenuation of lysosomal integrity measured by neutral red assay. An increase of reactive oxygen species (ROS) generation, DNA fragmentation, and phosphatidylserine externalization were quantified following cecropin A exposure at a concentration of 30 microM, whereas cecropin A-induced apoptosis was independent of caspase family members, because the activity of caspase-8 and -9 were irrelevant. Nevertheless, caspase-3 activity showed a significant increase at concentrations of 20-40 microM, but a considerable reduction at 50 microM. Flow cytometry analysis revealed a dissipation of the mitochondrial transmembrane potential (Deltapsi(m)), and the accumulation of cells at sub-G1 phase measured by FACS analysis of propidium iodide (PI) stained nuclei suggested induction of apoptosis. Morphological changes measured by Hoechst 33342 or acridine orange/ethidium bromide staining showed nuclear condensation, corroborating the apoptotic action of cecropin A. Overall, these data indicate that cecropin A is able to induce apoptosis in HL-60 cells through a signaling mechanism mediated by ROS, but independently of caspase activation., (Copyright 2010 Elsevier Inc. All rights reserved.)

Normal breast development is controlled by a balance between cell proliferation and apoptosis. The balance between the two parameters is crucial for determining the growth or regression of breast tumours in response to therapies and treatments. Therefore, it is necessary to understand the role of apoptosis in tumour progression. Active caspases participate as essential elements in the execution of apoptotic mechanisms. In the present study, we analysed the activities of caspase-3, -8 and -9 as well as cytochrome c release in N-methyl-nitrosourea (NMU)-induced rat mammary tumours, in order to establish the apoptotic events that occur in tumour growth in this animal model. Forty female virgin Wistar rats were randomly divided into two groups. One group was injected intraperitoneally with three doses of 50 mg/kg body weight of NMU. The control group received the vehicle only. After 122 days of NMU injection, the rats were sacrificed and the tumours were excised and processed. Results showed that in mammary tumours induced by NMU, the apoptotic death receptor-mediated pathway is activated through caspase-3 and -8, but the apoptotic mitochondrial pathway is suppressed through a non-activating process of caspase-9 activity, despite the release of cytochrome c. In conclusion, these findings have demonstrated a suppression of the apoptotic mitochondrial pathway through a non-activating process of caspase-9 activity, despite the release of cytochrome c in mammary tumours induced by NMU. Although the apoptotic death receptor-mediated pathway is activated, it is not enough to maintain the balance between proliferation and apoptosis, and thus determine the overall growth of the tumour.

The potential action of certain fatty acids has been studied since the early 1970s. Numerous effects on immune system functions have been related to dietary lipid administration; therefore, several of them have been applied in the treatment of inflammatory disorders. Nevertheless, n-3 polyunsaturated fatty acids may affect host resistance to infectious diseases. In addition, several studies have demonstrated that certain fatty acids are involved in apoptosis induction. Here, we have examined the action of different dietary lipids on the promotion of apoptosis in thymocytes from mice fed with dietary lipids and infected with Listeria monocytogenes. Thus, L. monocytogenes promoted an important cytotoxic effect in all of the groups, but it did not increase the percentage of DNA fragmentation. Similarly, an important increase of caspase-3 activity was demonstrated in OO and FO groups, but infection with L. monocytogenes exerted an inhibitory effect. Finally, L. monocytogenes did not modify proteasome activity among groups fed with dietary lipids. On the basis of this preliminary study, we can state that the infection of thymocytes from mice fed with dietary lipids does not promote a synergistic effect in the induction of apoptosis. Hence, these results may partially serve to elucidate the immune mechanisms involved in cells from mice fed with dietary lipids in an infectious process.

Background: The involvement of certain fatty acids in the induction of apoptosis has been established recently. In fact, considerable attention has been given in the past few years to the participation of polyunsaturated fatty acids as substances capable of modulating tumor cell growth., Materials and Methods: Fatty acids such as eicosapentaenoic acid (EPA), linolenic acid (LNA), arachidonic acid (AA), linoleic acid (LA), oleic acid (OA) or stearic acid (SA) were added to YAC-1 tumor cells., Results: Incubation of cells with fatty acids revealed a loss of cell viability in a dose-dependent manner. Quantification of DNA fragmentation showed a significant increase particularly in cells treated in the presence of LA, whereas the accumulation of triacylglycerols in the form of cytoplasmic lipid droplets was significantly enhanced in cells cultured with EPA, LNA or AA. The production of reactive oxygen species (ROS) was substantially increased after cell incubation. Nevertheless, the analysis of caspase-3 activity indicated a relevant increase in cells cultured in the presence of LA, OA or SA, but not in cells cultured with EPA, LNA or AA., Conclusion: On the basis of these results, we can speculate that long-chain polyunsaturated fatty acids such as EPA and AA as well as LNA induce cell death in YAC-1 lymphoma by an independent mechanism of caspase-3 activation.

Mitochondrial membrane permeabilization is a critical event in the process leading to physiological or chemotherapy-induced apoptosis. This permeabilization event is at least in part under the control of the permeability transition pore complex (PTPC), which interacts with oncoproteins from the Bcl-2 family as well as with tumor suppressor proteins from the Bax family, which inhibit or facilitate membrane permeabilization, respectively. Here we show that thiol crosslinking agents including diazenedicarboxylic acid bis 5N, N-dimethylamide (diamide), dithiodipyridine (DTDP), or bis-maleimido-hexane (BMH) can act on the adenine nucleotide translocator (ANT), one of the proteins within the PTPC. ANT alone reconstituted into artificial lipid bilayers suffices to confer a membrane permeabilization response to thiol crosslinking agents. Diamide, DTDP, and BMH but not tert-butylhydroperoxide or arsenite cause the oxidation of a critical cysteine residue (Cys 56) of ANT. Thiol modification within ANT is observed in intact cells, isolated mitochondria, and purified ANT. Recombinant Bcl-2 fails to prevent thiol modification of ANT. Concomitantly, a series of different thiol crosslinking agents (diamide, DTDP, and BMH, phenylarsine oxide) but not tert-butylhydroperoxide or arsenite induce mitochondrial membrane permeabilization and cell death irrespective of the expression level of Bcl-2. These data indicate that thiol crosslinkers cause a covalent modification of ANT which, beyond any control by Bcl-2, leads to mitochondrial membrane permeabilization and cell death.

Apoptosis is accompanied by major changes in ion compartmentalization and transmembrane potentials. Thymocyte apoptosis is characterized by an early dissipation of the mitochondrial transmembrane potential, with transient mitochondrial swelling and a subsequent loss of plasma membrane potential (DeltaP sip) related to the loss of cytosolic K+, cellular shrinkage, and DNA fragmentation. Thus, a gross perturbation of DeltaPsip occurs at the postmitochondrial stage of apoptosis. Unexpectedly, we found that blockade of plasma membrane K+ channels by tetrapentylammonium (TPA), which leads to a DeltaP sip collapse, can prevent the thymocyte apoptosis induced by exposure to the glucocorticoid receptor agonist dexamethasone, the topoisomerase inhibitor etoposide, gamma-irradiation, or ceramide. The TPA-mediated protective effect extends to all features of apoptosis, including dissipation of the mitochondrial transmembrane potential, loss of cytosolic K+, phosphatidylserine exposure on the cell surface, chromatin condensation, as well as caspase and endonuclease activation. In strict contrast, TPA is an ineffective inhibitor when cell death is induced by the potassium ionophore valinomycin, the specific mitochondrial benzodiazepine ligand PK11195, or by primary caspase activation by Fas/CD95 cross-linking. These results underline the importance of K+ channels for the regulation of some but not all pathways leading to thymocyte apoptosis.