Alpha-fetoprotein stimulates leukotriene synthesis in P388D1 macrophages

Alpha-fetoprotein (AFP) is able to bind specifically polyunsaturated fatty acids, especially arachidonic acid, the major precursor for prostaglandin and leukotriene synthesis. In P388D1 macrophages, AFP was found to reduce prostaglandin synthesis. This reduced synthesis was counter-balanced by a higher release of unmetabolized arachidonic acid and an enhanced production of leukotrienes. The same results were obtained with unactivated and activated cells irrespective of the activator used: lipopolysaccharide, Ca2+ ionophore A23187, phorbol myristate acetate, interferon-gamma, silica, or zymozan particles. The stimulation of leukotriene synthesis by AFP in macrophages thus appears to be a possible mechanism for the in vitro immunosuppressive effects of this oncofetal protein.     

The Parkinson-associated human P5B-ATPase ATP13A2 protects against the iron-induced cytotoxicity

P-type ion pumps are membrane transporters that have been classified into five subfamilies termed P₁–P₅. The ion transported by the P₅-ATPases is not known. Five genes named ATP13A1–ATP13A5 that belong to the P₅-ATPase group are present in humans. Loss-of-function mutations in the ATP13A2 gene (PARK9, OMIM 610513) underlay a form of Parkinson's disease (PD) known as the Kufor–Rakeb syndrome (KRS), which belongs to the group of syndromes of neurodegeneration with brain iron accumulation (NBIA).Here we report that the cytotoxicity induced by iron exposure was two-fold reduced in CHO cells stably expressing the ATP13A2 recombinant protein (ATP13A2). Moreover, the iron content in ATP13A2 cells was lower than control cells stably expressing an inactive mutant of ATP13A2. ATP13A2 expression caused an enlargement of lysosomes and late endosomes. ATP13A2 cells exhibited a reduced iron-induced lysosome membrane permeabilization (LMP). These results suggest that ATP13A2 overexpression improves the lysosome membrane integrity and protects against the iron-induced cell damage.     

Steroids and related products. LIV. The synthesis of 11-oxa steroids. VI. The synthesis of 11-oxatestosterone

The synthesis of 11-oxatestosterone from 11-oxa-5 alpha-androstane-3,17-dione, which is available from hecogenin, is described. The product shows, in comparison with the natural hormone, diminished androgenic and anabolic activities.     

Effects of naringin on cytosine arabinoside (Ara-C)-induced cytotoxicity and apoptosis in P388 cells

Naringin (NG), a flavonoid in grapefruit and citrus, has been reported to exhibit antioxidant effects and pharmacological actions. Recently, we have reported that NG suppressed the cytotoxicity and apoptosis induced by H(2)O(2), a typical pro-oxidant, in mouse leukemia P388 cells. Cytosine arabinoside (1-beta-d-arabinofuranosylcytosine; Ara-C) is the most important antimetabolite chemotherapeutic drug used for acute leukemia. It has been suggested that Ara-C-induced cytotoxicity is caused by apoptosis, which is mediated by reactive oxygen species (ROS). In this study, we examined the effect of NG on the cytotoxicity and apoptosis in mouse leukemia P388 cells treated with Ara-C. Ara-C caused cytotoxicity in a concentration and time-dependent manner in the cells. N-Acetyl-L-cysteine (NAC), cystamine (CysA) or a reduced form of glutathione (GSH), typical antioxidants significantly blocked Ara-C-induced cytotoxicity. Similarly, Ara-C-induced cell death was completely prevented by NG. NG strongly reduced ROS production caused by Ara-C in the cells. NG slightly increased the activities of antioxidant enzymes, catalase and glutathione peroxidase. Ara-C caused apoptosis with nuclear morphological change and DNA fragmentation. NG remarkably attenuated the Ara-C-induced apoptosis. NG completely blocked the DNA damage caused by Ara-C treatment at 6 h using the Comet assay. Our data suggest that NG reduces Ara-C-induced oxidative stress through both an inhibition of the generation of ROS production and an increase in antioxidant enzyme activities. Consequently, NG blocked apoptosis caused by Ara-C-induced oxidative stress, resulting in the inhibition of the cytotoxicity of Ara-C.     

Steroids and related products. LIII. The synthesis of 11-oxa steroids. V. The synthesis of 17-ethynyl-11-oxatestosterone

The synthesis of 17-ethynyl-11-oxatestosterone, both from 11-oxa-5 alpha-pregnane-3,20-dione and, via a 3,17-dioxygenated 9-oxo 9,12-seco 11-nor 5 alpha-androstane-12-oic ester, from 3 beta-acetoxy-17-hydroxy-5 alpha-pregnan-12-one--two products available from hecogenin--is reported. The new hormone analogue shows significant progestational activity in the Clauberg test and relatively weak activity in a post-coital antifertility assay.     

The chemical synthesis and cytotoxicity of new sulfur analogues of 2-methoxy-lysophosphatidylcholine

The chemical synthesis of phosphorothioate/phosphorodithioate analogues of 2-methoxy-lysophosphatidylcholine has been described. For the preparation of new sulfur derivatives of lysophosphatidylcholine both oxathiaphospholane and dithiaphospholane approaches have been employed. Each lysophospholipid analogue was synthesized as a series of five compounds, bearing different fatty acid residues both saturated (12:0, 14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1→2 acyl migration. The cellular toxicity of newly synthesized 2-methoxy-lysophosphatidylcholine derivatives was measured using MTT viability assay and lactate dehydrogenase release method.     

5-Alkynyl-2'-deoxyuridines: chromatography-free synthesis and cytotoxicity evaluation against human breast cancer cells

Starting with 5-iodo-2'-deoxyuridine, a series of 5-alkynyl-2'-deoxyuridines (with n-propyl, cyclopropyl, 1-hydroxycyclohexyl, p-tolyl, p-tert-butylphenyl, p-pentylphenyl, and trimethylsilyl alkyne substituents) have been synthesized via the palladium-catalyzed (Sonogashira) coupling reaction followed by a simplified isolation protocol (76-94% yield). The cytotoxic activity of modified nucleosides against MCF-7 and MDA-MB-231 human breast cancer cells has been determined in vitro. 5-Ethynyl-2'-deoxyuridine, the only nucleoside in the series containing a terminal acetylene, is the most potent inhibitor with IC(50) (microM) 0.4+/-0.3 for MCF-7 and 4.4+/-0.4 for MDA-MB-231.     

Fc gamma 2b receptor-mediated prostaglandin synthesis by a murine macrophage cell line (P388D1)

The nature of signals transmitted by two types of Fc gamma receptors (one specific for IgG2b and the other for IgG2a) present on the surface of a murine macrophage cell line (P388D1) was investigated. Specific binding of IgG2b (presented as EA2b) to cell surface Fc gamma 2br triggered the release of 3H-arachidonic acid and 3H-prostaglandins (PG) from P388D1 cells that were prelabeled with 3H-arachidonate. The release of 3H-arachidonic acid, which increased in a dose-dependent manner, was enhanced by exogenous Ca++ (1.25 mM) and was completely blocked by ethylenediaminetetraacetate (EDTA) (4 mM) or a phospholipase A2 inhibitor, p-bromophenacylbromide (7 microgram/ml). A cyclooxygenase inhibitor, indomethacin (9 microgram/ml), reduced the 3H-arachidonic acid release and completely blocked the conversion of arachidonate into PG. Cytochalasin D (1 microgram/ml), which inhibited the phagocytosis of immune complexes by 90% of P388D1 cells, did not affect the Fc gamma 2bR-triggered release of arachidonic acid. Specific binding of IgG2a (presented as EA2a) to cell surface Fc gamma 2aR did not trigger the release of either 3H-arachidonic acid or 3H-PG from P388D1 cells. Our data demonstrate a signal for the activation of the arachidonic acid metabolic cascade is transmitted by Fc gamma 2bR, but not by Fc gamma 2aR, on the surface of P388D1 cells, probably through the initial activation of the phospholipase A2 activity associated with Fc gamma 2bR.     

Effects of cis-unsaturated fatty acids on doxorubicin sensitivity in P388/DOX resistant and P388 parental cell lines

It has been reported that several cis-unsaturated fatty acids (c-UFAs) could increase doxorubicin (DOX) accumulation in cancer cells and hence elevate its cytotoxicity. However, some researchers showed that c-UFA pretreatment did not affect its cytotoxicity in special cell lines. It is possible that the different results occurred due to different cellular characteristics. We hypothesized that c-UFA treatment might modulate the activities of some antioxidant enzymes to affect the resistance of cells to DOX. In the present study, we examined how c-UFA pretreatment affected DOX cytotoxicity on mouse leukemia cell line, P388, and its resistant subline, P388/DOX, which we found to have significantly higher glutathione peroxidase (GPx) activity as well as P-glycoprotein (p-gp) overexpression. We chose two c-UFAs, gamma-linolenic acid (GLA) (18:3n-6) and docosahexaenoic acid (DHA) (22:6n-3). Cytotoxicity was measured by MTT (3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and trypan blue exclusion assays. DOX accumulation and p-gp expression were measured by flow cytometry. The activities of catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and GPx were determined for both cell lines with and without treatment with GLA or DHA. Significant DOX accumulation occurred in both cell lines with GLA or DHA pretreatment, but without any change in p-gp expression in either cell line. Sensitivity to DOX cytotoxicity was improved by GLA or DHA pretreatment in P388/DOX in which only SOD activity was significantly increased, but not in the parental cell line P388 in which both SOD and CAT were significantly increased by the pretreatment. However, combined pretreatment of GLA or DHA with antioxidants, pyrrolidinedithiocarbamate (PDTC) or Vitamin C, could sensitize not only P388/DOX but also P388 cells to DOX. We conclude that the effects of c-UFA pretreatment on the sensitivity of cancer cells to DOX not only depend on the change in drug accumulation but also the change in the levels of antioxidant enzyme activities, and suggest that combined administration of c-UFAs, antioxidants, and DOX may be more effective in treating leukemia.     

Differences in myristic acid synthesis and in metabolic rate for P388 cells resistant to doxorubicin

Lipids extracted from doxorubicin-resistant murine leukemia cells (P388/ADR) contained greater relative amounts of myristic and palmitoleic acids than lipids from sensitive cells (P388). This was seen in both the phospholipid and neutral lipid fractions under two nutritional conditions. Correspondingly in P388/ADR cells, myristic acid comprised a greater proportion of the products of the fatty acid synthetase system, and acyl-CoA 9-desaturase activity was transiently greater than in P388. Similar alterations in myristic acid synthesis were exhibited by DC3F/AD X, N417/VP-16, and P388/AZQ30U cells but not by CHRC5 or HL60/AR cells. This alterations was independent of alterations in the P180 glycoprotein and might be linked via the myristoylation of proteins to a different mechanism of drug resistance. Doxorubicin-resistant P388/ADR cells also exhibited a much higher rate of oxidative energy production.     

The effect of 3,4-disuccinyldianhydrogalactitol, N-nitrosourea and their combination on DNA synthesis in normal and mouse P388 tumor cells

The rates of incorporation of 2-14C-thymidine into DNA of leukemia P388, bone marrow, gastrointestinal mucosa and spleen cells at various time after administration of 3,4-disuccinyldianhydrogalactitol (DisuDAG), 1-methyl-1-nitrosourea (MNU), 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea (HECNU) and their combinations at different doses to mice with leukemia P388 (solid form) were studied. DisuDAG (80 mg/kg) induced the deep and the stable inhibition in DNA synthesis of leukemia P388, bone marrow and spleen cells. The combination of DisuDAG and HECNU at small doses induced the deep and the stable suppression of DNA synthesis in tumor cells, however DNA synthesis in normal dividing cells was shown to recover more rapidly than in leukemia P388 cells. Administration of the combination of DisuDAG with MNU to tumor-bearing mice induced more stable inhibition of DNA synthesis in tumor cells in comparison with MNU and DisuDAG. In vivo inhibition of DNA synthesis in leukemia P388 cells with DisuDAG and HECNU was not due to damage in pool of precursors (TCA soluble fraction).     

The beta-VLDL receptor pathway of murine P388D1 macrophages

Very low density lipoproteins Sf 100-400 (VLDL1) from hypertriglyceridemic (HTG) subjects and chylomicrons cause receptor-mediated lipid engorgement in unstimulated macrophages in vitro via the beta-VLDL receptor pathway. We now report that the murine macrophage P388D1 cell line possesses the characteristics of the beta-VLDL receptor pathway observed previously in freshly isolated resident murine peritoneal macrophages or human monocyte-macrophages. HTG-VLDL1 isolated from the plasma of subjects with hypertriglyceridemia types 3, 4, and 5 interact with P388D1 macrophages in a high-affinity, curvilinear manner. beta-VLDL, HTG-VLDL1, chylomicrons, and thrombin-treated HTG-VLDL1 (which do not bind to the LDL receptor) compete efficiently and similarly for the uptake and degradation of HTG-VLDL1. LDL and acetyl LDL do not compete, indicating that uptake of HTG-VLDL1 is via neither the LDL receptor nor the acetyl LDL receptor. Binding of thrombin-treated HTG-VLDL1 to the beta-VLDL receptor indicates that the thrombin-accessible apoE, which is absolutely required for interaction of HTG-VLDL Sf greater than 60 with the LDL receptor, is not required for binding to the beta-VLDL receptor. The uptake and degradation of 125I-labeled HTG-VLDL1 is suppressed up to 80-90% by preincubation of the cells with sterols, acetyl LDL, or beta-VLDL, indicating that this process is not via the irrepressible chylomicron remnant (apoE) receptor. Chylomicrons, HTG-VLDL1, and thrombin-treated HTG-VLDL1-but not normal VLDL1, beta-VLDL, LDL, or acetyl LDL-produce massive triglyceride accumulation (10-20-fold mass increases in 4 hr) in P388D1 macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antitrypanosomal activities and cytotoxicity of 5-nitro-2-furancarbohydrazides

A series of 5-nitro-2-furancarbohydrazides were synthesized. In vitro antitrypanosomal activities of these compounds were determined against the closely related protozoa Trypanosoma cruzi Trypanosoma brucei and discussed in relation to potential targets.     

Isolation of highly multidrug-resistant P388 cells from drug-sensitive P388/S cells by flow cytometric cell sorting

To investigate the spontaneous frequency of occurrence of stable multidrug-resistant cells in a population of drug-sensitive cells, we exposed drug sensitive P388/S cells to daunorubicin (dnr) for 1 h, then used fluorescence-activated cell sorting based on intracellular dnr fluorescence to isolate cells within P388/S having different intracellular content of drug. One of the sort windows chosen (low dnr content sort window) isolated only P388/S cells with intracellular drug content equal to or less than that of the known multidrug-resistant subline P388/adr. This sort window constituted approximately 3% of P388/S cells with lowest dnr content. By such a procedure we were able, on one of seven attempts, to isolate and cultivate stable, highly multidrug-resistant cells (comparable to that of P388/adr) from the P388/S cells obtained from the low dnr-content sort window. Net growth of cells in culture was observed 15-20 days after sorting, indicating that of the P388/S cells collected from the low dnr-content sort window, very few were actually highly drug-resistant. On no occasion could resistant cells be cultivated from cells sorted from P388/S with higher dnr content, as would be expected if mutation to a multidrug-resistant phenotype had occurred as a result of exposure to drug. The resistant cells isolated from P388/S by sorting (called P388/LoSort) displayed low intracellular accumulation of dnr that was enhanced by verapamil, were cross-resistant to vincristine and actinomycin-D, and distinct from P388/S, possessed a 150- to 160-kD membrane species identified by Vinca alkaloid photoaffinity labeling.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro cytotoxicity of Tanshinones isolated from Salvia miltiorrhiza Bunge against P388 lymphocytic leukemia cells

The cytotoxic effect of four tanshinones isolated from the dried root of Salvia miltiorrhiza Bunge (Lamiaceae) were studied in vitro using P388 lymphocytic leukemia cells. Tanshinone I and tanshinone IIA were shown to be quite strongly cytotoxic against the cells (86.76% and 56.05% cell inhibition at 25 microg/ml). Dihydrotanshinone I and cryptotanshinone, which lack of saturation at C-15, showed little cytotoxicity (13.71% and 39.21% cell inhibition at 25 microg/ml). The results also revealed a structure activity relationship. We suggest that the unsaturation at C-15 and saturation on ring A may be critical structural components for cytotoxicity in these diterpenes.