M. Cristina Tejedor

Ganoderma lucidum induced apoptosis in NB4 human leukemia cells: Involvement of Akt and Erk

AIM OF THE STUDY The final goal of this work was to study the toxic and apoptosis effects induced by fractions from Ganoderma lucidum [Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk] on NB4 human leukemia cells. MATERIALS AND METHODS Two aqueous extracts and a methanol-extracted column-chromatography semipurified fraction were obtained from Ganoderma lucidum fruiting body. Flow cytometry analyses were used to measure cell viability, cell cycle and DNA fragmentation and to quantify apoptosis. Western-blot analyses were used to quantify changes in apoptosis proteins and intracellular kinases. RESULTS Aqueous extracts slightly reduce cell viability and induce DNA fragmentation in NB4 cells. Methanol-extracted semipurified fraction at dilutions down to 15% or 40% of the initial fraction concentration reduced significantly the viability of these leukemia cells (treated for 19h) with induction of DNA fragmentation and induction of apoptosis. Overmore, the dilution down to 15% of the initial E3 concentration induced a reduction of p53 levels, of the Bcl2/Bax relationship as well as reduced levels of both unphosphorylated and phosphorylated Akt (Protein kinase Akt, protein kinase B) and Erk (Erk1 and 2). CONCLUSIONS Induction of apoptosis and alterations in signal transduction kinases (Akt and Erk) are produced by active fractions from Ganoderma lucidum on human leukemia cells. These data could be of important relevance from the viewpoint of antitumor actions of compounds from Ganoderma lucidum. Eventual therapy applications in leukemia cells might be developed.

In vitro phagocytosis of carrier mouse red blood cells is increased by Band 3 cross-linking or diamide treatment

Chemical alteration of red blood cells (RBCs) can induce increased phagocytosis of modified cells by macrophages. In this study we have used different chemical treatments for the modification of the mouse red‐blood‐cell membrane surface, namely oxidant compounds, such as ascorbate/Fe+2 and diamide [azodicarboxylic acid bis(dimethylamide)], or Band 3‐cross‐linking reagents. We monitored the phagocytosis of oxidized or Band 3‐cross‐linked mouse red blood cells by peritoneal macrophages. The extent of phagocytosis of RBCs is not affected by oxidation with ascorbate/Fe3+, but it is increased (up to 10%) by oxidation with 2 mM diamide. Furthermore, phagocytosis is greatly increased (up to 40%) as a result of cross‐linking with either of two Band 3 bifunctional reagents [bis(sulphosuccinimidyl) suberate (BS3) and 3,3′‐dithiobis(sulphosuccinimidyl propionate) (DTSSP)]. To evaluate targeting towards macrophages of such modified RBCs for therapeutical purposes, we have determined the phagocytosis of Band 3 carrier RBCs loaded with carbonic anhydrase. In this case phagocytosis is high enough (25%) to deliver the enzyme into macrophages. We have also assayed the influence of serum components and IgG on the efficiency of phagocytosis and discuss the possible phagocytosis mechanisms. In the case of BS3‐cross‐linked carrier RBCs, phagocytosis is markedly enhanced (from 12% up to 25%) by serum components. This opens a way for therapeutic application of these carrier RBCs, with special relevance in short‐term delivery to cells of the mononuclear phagocytic system.

Partial purification of glucose 6-phosphate dehydrogenase and phosphofructokinase from rat erythrocyte haemolysate by partitioning in aqueous two-phase systems.

Glucose 6-phosphate dehydrogenase shows a high partition coefficient in poly-(ethylene glycol)-dextran aqueous two-phase systems in comparison with those for 6-phosphogluconate dehydrogenase, phosphofructokinase and the bulk of proteins present in rat erythrocyte haemolysates. As a consequence, fractions highly enriched in glucose 6-phosphate dehydrogenase can be obtained after multiple partitions in the above systems with a counter-current distribution procedure. Phosphofructokinase shows a high affinity for Cibacron Blue and, as a result, the enzyme can be extracted in the top phase of poly(ethylene glycol)-dextran systems containing Cibacron Blue-poly(ethylene glycol) (affinity systems). The efficiency for the purification of the enzymes by partitioning is increased up to 10-fold when enzyme-rich fractions, obtained by precipitation with poly(ethylene glycol), are used instead of original haemolysate. The recovery of enzyme activities is near 100% in both instances.

Delivery to macrophages of interleukin 3 loaded in mouse erythrocytes.

Mouse carrier erythrocytes containing 125I-interleukin 3 have been prepared and treated with band 3 crosslinking reagents. The incorporation of interleukin 3 by hypotonic treatment into mouse erythrocytes reached levels of about 15% of the interleukin 3 added to the medium being predominantly present in the cytosolic fraction (73%). Uptake fell to about 7.4% when using the same conditions but omitting hypotonic shock. The interaction of band 3 crosslinked interleukin 3 loaded erythrocytes with macrophages was also studied. A high level of incorporation of interleukin 3 into macrophages was observed either from band 3 crosslinked, interleukin 3-loaded erythrocytes or from interleukin 3 loaded erythrocytes. The observations encourage the view that the system may be able to deliver and target cytokines and other growth factors to macrophages.

CYTOTOXIC ACTION OF GANODERMA LUCIDUM ON INTERLEUKIN-3 DEPENDENT LYMPHOMA DA-1 CELLS: INVOLVEMENT OF APOPTOSIS PROTEINS

Aqueous extracts and a semipurified fraction obtained by methanol extraction and column chromatography were isolated from Ganoderma lucidum [Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk] and their effects on interleukin 3‐dependent lymphoma cells (DA‐1) were studied. Cell viability was reduced by the action of unboiled aqueous extract and by the methanol‐extracted column‐chromatography semipurified fraction, producing DNA fragmentation in DA‐1 cells. Treatments with aqueous extracts showed increments of Bax after 13 h, increments of p53 and Mdm2 after 19 h and a reduction of these three proteins after 24 h. The methanol‐extracted semipurified fraction also induced increments of p53 and Mdm2 factors at 19 h with a reduction after 24 h. The methanol‐extracted column‐chromatography semipurified fraction from Ganoderma lucidum produced minor changes in the level of Akt after treatments for 19 h in DA‐1 cells with a slight reduction in the levels of NFkB‐p65 factor. Both the unboiled aqueous extract and the methanol‐extracted column‐chromatography semipurified fraction produced cleavage of inactive caspase 3, as a clear indication of induction of apoptosis by compounds present in Ganoderma lucidum. Copyright

Differential In vitro and In vivo Behavior of Mouse Ascorbate/Fe3+ and Diamide Oxidized Erythrocytes

Chemical oxidation of mouse erythrocytes has been carried out using two different oxidizing systems namely: Diamide and Ascorbate/Fe3+ together with different concentrations of the oxidant. These oxidation treatments produced different extents of modification in membrane proteins as was observed by electrophoretic analyses that showed a possible formation of high molecular weight aggregates. Lipid peroxidation was also observed as the result of these chemical treatments. The action of these two oxidation treatments produced different extents of lipid peroxidation in which the effect Ascorbate/Fe3+ reached higher values than that shown by diamide treatments. To study the resulting in vitro behavior of such oxidized erythrocytes, we have evaluated the recognition of oxidized erythrocytes by peritoneal macrophages. In the conditions used, diamide oxidized erythrocytes were more highly recognized by macrophages than Ascorbate/Fe3+ treated erythrocytes. However, in both cases an influence of serum factors in the recognition process can be inferred. Additionally, we have correlated on one side the action of different oxidation systems on mouse erythrocytes with different in vivo behavior and organ uptake of the oxidized erythrocytes. On the other side, differential targeting of oxidized erythrocytes to a liver or spleen was observed on dependence of the oxidant used.

Heterogeneity of hypotonically loaded rat erythrocyte populations as detected by counter-current distribution in aqueous polymer two-phase systems

Carrier rat erythrocytes loaded with exogenous substances ([125I] carbonic anhydrase) by hypotonic-isotonic dialysis become heterogeneous cell populations that can be fractionated using the counter-current distribution (CCD) technique. Two well-defined low- and high-partition ratio, G, subpopulations are obtained in charge-sensitive dextran-polyethylene glycol two-phase systems. The low-G subpopulation, which contains the most fragile and surface-altered cells, as deduced from their osmotic fragility curves and partition behaviour, respectively, presents a high amount of exogenous substance incorporated (134.6 cpm/10(6) cells). The high-G subpopulation, that contains cells similar to the control or isotonically dialyzed cells presents a lower amount of exogenous substance incorporated (69.8 cpm/10(6) cells). Cells in this high-G subpopulation seem to be fractionated, like the controls, according to ageing as suggested by the decline of the pyruvate kinase specific activity from the left- to the right-hand side of the CCD profile.

Affinity partitioning of erythrocytic phosphofructokinase in aqueous two-phase systems containing poly(ethylene glycol)-bound Cibacron Blue influence of pH, ionic strength and substrates/effectors

Phosphofructokinase (PFK) from rat erythrocyte haemolysates has a high affinity for Cibacron Blue F3G-A covalently bound to poly(ethylene glycol) (PEG-Cb) and thus the enzyme can be extracted into the top phase of poly(ethylene glycol)-dextran aqueous two-phase systems containing PEG-Cb. The pH, ionic strength and presence of substrates/effectors affect to different extents the affinity of the enzyme for PEG-Cb and the number of PEG-Cb molecules attached per molecule of PFK (the latter probably reflecting, at saturation, the influence on the aggregation state of the enzyme) and thus influence the yield of enzyme recovered in the top phase. Increasing the pH from 6 to 7 and then to 8 leads to a higher yield of PFK in the top phase. A change in pH from 6 to 7 and 8 results in an increased number of PEG-Cb molecules attached per molecule of enzyme while the affinity of PFK for PEG-Cb shows a minimum at pH 7. The ionic strength in the range 0.017-0.164 has less influence on the partitioning of PFK. The presence of substrates or effectors of the enzyme in general reduces the recovery of PFK in the top phase. Fructose 6-phosphate increases the number of PEG-Cb molecules attached but greatly reduces the affinity of PFK for PEG-Cb. In contrast, AMP slightly reduces the number of PEG-Cb molecules attached and the affinity of PFK for PEG-Cb. ATP and ATP-Mg2+ compete with PEG-Cb for the same binding sites in PFK.(ABSTRACT TRUNCATED AT 250 WORDS)

Band‐3 crosslinking‐induced targeting of mouse carrier erythrocytes

Mouse band‐3 crosslinked carrier erythrocytes have been prepared. [125I]Carbonic anhydrase (CA) has been encapsulated into mouse erythrocytes. Then, loaded erythrocytes were labelled with 51Cr. Eventually, these doubly labelled cells were crosslinked with band‐3 crosslinking reagents. [125I]CA was shown to have cytosolic localization in crosslinked carrier erythrocytes. Estimation of the action of band‐3 crosslinkers on mouse carrier‐erythrocyte membranes rendered values around 17–21% of band‐3 monomer reduction. Crosslinked carrier erythrocytes were in vivo targeted to liver, as shown by chromium‐labelling localization. Also, encapsulated CA radioactivity was localized in vivo predominantly in liver, which is clearly in contrast with the behaviour shown by free CA injected into animals. These results support this model as a feasible system for the analysis of carrier‐erythrocyte survival and targeting as well as the in vivo efficacy of release and targeting of encapsulated compounds.

JNK and NFκB dependence of apoptosis induced by vinblastine in human acute promyelocytic leukaemia cells

The relationship between the mitogen‐activated protein kinase response, nuclear factor‐κB (NFκB) expression and the apoptosis in human acute promyelocytic leukaemia NB4 cells treated with vinblastine was investigated in this work. Cell viability, subdiploid DNA and cell cycle were analysed by propidium iodide permeability and flow cytometry analyses. Apoptosis was determined by annexin V‐Fluorescein isothiocyanate assays. Western‐blot analysis was used for determination of expression levels of apoptotic factors (p53, Bax and Bcl2), intracellular kinases [serine/threonine‐specific protein kinase, extracellular signal‐regulated kinase and c‐Jun N‐terminal kinase (JNK)], NFκB factor and caspases. Electrophoretic mobility shift assay was usefully applied to study DNA‐NFκB interaction. In NB4 cells, vinblastine produces alteration of p53 and DNA fragmentation. Vinblastine treatment had an antiproliferative effect via the induction of apoptosis producing Bax/Bcl‐2 imbalance. Vinblastine treatment suppressed NFκB expression and depressed NFκB‐DNA binding activity while maintaining JNK activation that subsequently resulted in apoptotic response through caspase‐dependent pathway. Our study provides a possible anti‐cancer mechanism of vinblastine action on NB4 cells by deregulation of the intracellular signalling cascade affecting to JNK activation and NFκB expression. Moreover, JNK activation and NFκB depression can be very significant factors in apoptosis induction by vinblastine. Copyright