Early Phosphatidylserine Exposure Research Articles

Acyclic Sesquiterpenes from the Fruit Pericarp of Sapindus saponaria Induce Ultrastructural Alterations and Cell Death in Leishmania amazonensis.

Previous studies reported antiprotozoal activities of Sapindus saponaria L. The aim of this work was the evaluation of antileishmanial activity and mechanism of action of extract and fractions of S. saponaria L. Hydroethanolic extract (EHA) obtained from fruit pericarps was fractionated using solid-phase extraction in a reversed phase, resulting in fractions enriched with saponins (SAP fraction) and acyclic sesquiterpene oligoglycosides (OGSA fraction). The activities of EHA, SAP, and OGSA were evaluated by antiproliferative assays with promastigote and intracellular amastigote forms. Cytotoxicity on macrophages and hemolytic activity were also analyzed. Morphological and ultrastructural changes in Leishmania amazonensis promastigotes were evaluated by electron microscopy. Flow cytometry was used to investigate mitochondrial dysfunction and phosphatidylserine exposure. OGSA was more selective for parasites than mammalian J774A1 macrophage cells, with selectivity indices of 3.79 and 7.35, respectively. Our results showed that only the OGSA fraction did not present hemolytic activity at its IC50 for promastigote growth. Electron microscopy revealed changes in parasite flagellum, cell body shape, and organelle size, mainly mitochondria. Flow cytometry analysis indicated mitochondrial membrane and cell membrane dysfunction. OGSA showed antileishmanial activity, resulting in several changes to protozoa cells, including mitochondrial depolarization and early phosphatidylserine exposure, suggesting a possible apoptotic induction.

Synthesis of thiophene-thiosemicarbazone derivatives and evaluation of their in vitro and in vivo antitumor activities

A series of thiophene-2-thiosemicarbazones derivatives (5–14) was synthesized, characterized and evaluated for their antitumor activity. They were tested in vitro against human tumor cell lines through the colorimetric method. The results revealed that compounds 7 and 9 were the most effective in inhibiting 50% of the cell growth after 48 h of treatment. As compound 7 showed a potent antiproliferative profile, it has been chosen for further studies in 786-0 cell line by flow cytometry. Treatments with compound 7 (50 μM) induced early phosphatidylserine exposure after 18 h of exposure and this process progressed phosphatidylserine exposure with loss of cell membrane integrity after 24 h of treatment, suggesting a time-dependent cell death process. Regarding the cell cycle profile, no changes were observed after treatment with compound 7 (25 μM), suggesting a mechanism of cell death independent on the cell cycle. The in vivo studies show that compound 7 possess low acute toxicity, being the doses of 30–300 mgKg−1 chosen for studies in Ehrlich solid tumor model in mice. All doses were able to inhibit tumor development being the lowest one the most effective. Our findings highlight thiophene-2-thiosemicarbazones as a promising class of compounds for further studies concerning new anticancer therapies.

Enhanced Tolerance against Early and Late Apoptotic Oxidative Stress in Mammalian Neurons through Nicotinamidase and Sirtuin Mediated Pathways

Focus upon therapeutic strategies that intersect between pathways that govern cellular metabolism and cellular survival may offer the greatest impact for the treatment of a number of neurodegenerative and metabolic disorders, such as diabetes mellitus. In this regard, we investigated the role of a Drosophila nicotinamidase (DN) in mammalian SH-SY5Y neuronal cells during oxidative stress. We demonstrate that during free radical exposure to nitric oxide generators DN neuronal expression significantly increased cell survival and blocked cellular membrane injury. Furthermore, DN neuronal expression prevented both apoptotic late DNA degradation and early phosphatidylserine exposure that may serve to modulate inflammatory cell activation in vivo. Nicotinamidase activity that limited nicotinamide cellular concentrations appeared to be necessary for DN neuroprotection, since application of progressive nicotinamide concentrations could abrogate the benefits of DN expression during oxidative stress. Pathways that involved sirtuin activation and SIRT1 were suggested to be vital, at least in part, for DN to confer protection through a series of studies. First, application of resveratrol increased cell survival during oxidative stress either alone or in conjunction with the expression of DN to a similar degree, suggesting that DN may rely upon SIRT1 activation to foster neuronal protection. Second, the overexpression of either SIRT1 or DN in neurons prevented apoptotic injury specifically in neurons expressing these proteins during oxidative stress, advancing the premise that DN and SIRT1 may employ similar pathways for neuronal protection. Third, inhibition of sirtuin activity with sirtinol was detrimental to neuronal survival during oxidative stress and prevented neuronal protection during overexpression of DN or SIRT1, further supporting that SIRT1 activity may be necessary for DN neuroprotection during oxidative stress. Implementation of further work to elucidate the cellular mechanisms that govern nicotinamidase activity in mammalian cells may offer novel avenues for the treatment of disorders tied to oxidative stress and cellular metabolic dysfunction.

Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death

Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death and marks those cells for clearance by macrophages. Macrophages secrete lactadherin, a PS-binding protein, which tethers apoptotic cells to macrophage integrins. We utilized fluorescein-labeled lactadherin together with the benchmark PS Probe, annexin V, to detect PS exposure by flow cytometry and confocal microscopy. Immortalized leukemia cells were treated with etoposide, and the kinetics and topology of PS exposure were followed over the course of apoptosis. Costaining etoposide-treated leukemoid cells with lactadherin and annexin V indicated progressive PS exposure with dim, intermediate, and bright staining. Confocal microscopy revealed localized plasma membrane staining, then diffuse dim staining by lactadherin prior to bright generalized staining with both proteins. Annexin V was primarily localized to internal cell bodies at early stages but stained the plasma membrane at the late stage. Calibration studies suggested a PS content less, less than or approximately equal to 2.5%-8% for the membrane domains that stained with lactadherin but not annexin V. Macrophages may utilize lactadherin to detect PS exposure prior to exposure of sufficient PS to bind annexin V. The methodology enables detection of PS exposure at earlier stages than established methodology.

Involvement of Sodium in Early Phosphatidylserine Exposure and Phospholipid Scrambling Induced by P2X7 Purinoceptor Activation in Thymocytes

Extracellular ATP (ATP(ec)), a possible effector in thymocyte selection, induces thymocyte death via purinoceptor activation. We show that ATP(ec) induced cell death by apoptosis, rather than lysis, and early phosphatidylserine (PS) exposure and phospholipid scrambling in a limited thymocyte population (35-40%). PS externalization resulted from the activation of the cationic channel P2X7 (formerly P2Z) receptor and was triggered in all thymocyte subsets although to different proportions in each one. Phospholipid movement was dependent on ATP(ec)-induced Ca(2+) and/or Na(+) influx. At physiological external Na(+) concentration, without external Ca(2+), PS was exposed in all ATP(ec)-responsive cells. In contrast, without external Na(+), physiological external Ca(2+) concentration promoted a submaximal response. Altogether these data show that Na(+) influx plays a major role in the rapid PS exposure induced by P2X7 receptor activation in thymocytes.

Novel apoptosis-inducing trans-platinum piperidine derivatives: synthesis and biological characterization.

The synthesis, chemical characterization, and interaction with cells of new sterically hindered trans- and cis-diaminedichloroplatinum(II) complexes are described. The amine ligands include monofunctional piperidine (pip) and piperazine (pz). The poor solubility of trans-diaminedichloroplatinum complexes was overcome by introducing the positively charged pz ligand, which allows retaining of the classic platinum coordination sphere. In vitro evaluation in OV-1063 and C-26 tumor cells revealed that replacing one NH3 of the inactive transplatin by an aromatic planar ligand (4-picoline, 4-pic) or by an aliphatic nonplanar heterocyclic ligand (pip) or replacing both NH3 groups with a 4-pic ligand and a pip or pz ligand significantly increases the cytotoxic activity of these complexes. The unsymmetric complexes trans-[PtCl2(4-pic)(pip)] and trans-[PtCl2(4-pic)(pz)]HCl were the most cytotoxic compounds against the cisplatin-sensitive tumor cell line C-26 (IC50 = 4.5 and 5.5 microM, respectively) and the cisplatin-sensitive tumor cell line OV-1063 (IC50 = 6.5 and 7.4 microM, respectively). In contrast, replacing one NH3 of the cis isomer by an aromatic planar ligand (4-pic) or by an aliphatic amine lowered their cytotoxiciy in comparison to cisplatin. Cell penetration and Pt-DNA adduct formation were also evaluated, and it was clearly shown that both trans-[PtCl2(4-pic)(pip)] and trans-[PtCl2(4-pic)(pz)]HCl penetrate efficiently the cellular membrane of the tumor cells and platinate the cellular DNA. When comparing cellular DNA platination, positively charged trans-[PtCl2(4-pic)(pz)]HCl was 7-fold higher than both cisplatin and its neutral analogue trans-[PtCl2(4-pic)(pip)]. Moreover, in contrast to cisplatin, in the cell lines used, cell death caused by both complexes appeared to be apoptotic according to several criteria including early phosphatidylserine exposure, activation of caspases, and characteristic morphological changes. Our results suggest that these novel mixed nonclassical trans-Pt(II) complexes are biologically and mechanistically distinct from known Pt complexes and deserve evaluation of their efficacy in tumor-bearing animals.