Degranulating activity in hybrid cells derived from rat peritoneal mast cells and ehrlich ascites

Fusion of rat mast cells and Ehrlich ascites tumor cells was mediated by HVJ. Compound 48/80-induced degranulation occurred in the fused cells formed from two mast cells and one tumor cell, but not in the fused cells from one mast cell and two or more tumor cells.     

Stability of artemisinin in aqueous environments: impact on its cytotoxic action to Ehrlich ascites tumour cells

We have recently shown artemisinin to be cytotoxic against Ehrlich ascites tumour cells. The aim of this study was to investigate the stability of this compound in the aqueous environment of the in-vitro Ehrlich ascites tumour cell system (RPMI 1640 cell culture medium supplemented with 10% foetal bovine serum (RPMI/FBS) with reference to its cytotoxic action. Literature data show that artemisinin can react with Fe2+ yielding reactive intermediates leaving artemisinin G as a major end-product. The current study showed that only excess addition of Fe2+ to artemisinin in distilled water, phosphate-buffered saline (PBS) and RPMI/FBS and incubation for 24 h led to degradation of artemisinin and yielded artemisinin G. If Fe2+ was not added results from HPLC analysis were indicative of complete recovery of artemisinin from distilled water and RPMI/FBS, with or without cells, at 37 degrees C for at least 24 h. In addition, incubation of artemisinin in RPMI/FBS with or without cells at 37 degrees C for 24 h before cytotoxicity assay did not change its cytotoxic action. On the basis of these results, we suggest that cytotoxicity to tumour cells was caused by unchanged artemisinin. This is not so for the antimalarial activity of artemisinin and derivatives, for which the presence of a pool of (haem) Fe2+ is a prerequisite resulting in free radicals or electrophilic intermediates or both.     

Tea plant root extract (TRE) as an antineoplastic agent

The antitumour effect of tea plant root extract (TRE) has been evaluated against Ehrlich ascites carcinoma (EAC) in Balb-C mice. Significant increases of survival times of the TRE-treated, tumour-bearing mice have been confirmed repeatedly with respect to the control group. TRE inhibited the tumour cell growth and reversed the changes of haematological parameters consequent to tumour inoculation.     

Effect of age and sex on lung-colony-forming efficiency of injected mouse tumour cells

The i.v. injection of a specified number of cells of either an Ehrlich ascites tumour (ELD) or spontaneous mouse mammary adenocarcinomas (MA) into C3H mice yielded a number of lung colonies which varied significantly with the age or sex of recipient mice. The yield was higher in mice of 71 weeks than in those of 15 weeks, except for MA cells injected into females, when the yield was higher in the younger mice. Sex did not influence very significantly the yield of colonies from ELD cells; in the case of MA cells the direction of sex differences depended on age. A difference in the effect of pre-immunization with age was not observed.     

Cytotoxic and anticancer activities of some 1-aryl-2-dimethylaminomethyl-2-propen-1-one hydrochlorides

A series of 1-aryl-2-dimethylaminomethyl-2-propen-1-one hydrochlorides demonstrated marked cytotoxicity towards approximately 55 human tumour cell lines from different neoplastic diseases. In general they were more potent than melphalan and displayed selective toxicity towards human leukemic cells. A representative compound, 1-phenyl-2-dimethyl-aminomethyl-2-propen-1-one hydrochloride (2a), had similar cytotoxicity as melphalan towards murine P388 and L1210 leukemic cells. In addition, 2a reduced the sizes of a number of human tumour xenografts including colon, prostatic and melanotic cancers passaged in athymic mice. Compound 2a showed excellent activity towards Ehrlich ascites carcinoma and B16F1 melanoma in mice which was enhanced using niosomes. One may conclude from the data generated that 1-aryl-2-dimethylaminomethyl-2-propen-1-one hydrochlorides are a novel series of cytotoxic and anticancer agents.     

Changes in glutathione status and the antioxidant system in blood and in cancer cells associate with tumour growth in vivo

The relationship among cancer growth, the glutathione redox cycle and the antioxidant system was studied in blood and in tumour cells. During cancer growth, the glutathione redox status (GSH/GSSG) decreases in blood of Ehrlich ascites tumour-bearing mice. This effect is mainly due to an increase in GSSG levels. Two reasons may explain the increase in blood GSSG: (a) the increase in peroxide production by the tumour that, in addition to changes affecting the glutathione-related and the antioxidant enzyme activities, can lead to GSH oxidation within the red blood cells; and (b) an increase of GSSG release from different tissues into the blood. GSH and peroxide levels are higher in the tumour cells when they proliferate actively, however GSSG levels remain constant during tumour growth in mice. These changes associate with low levels of lipid peroxidation in plasma, blood and the tumour cells. The GSH/GSSG ratio in blood also decreases in patients bearing breast or colon cancers and, as it occurs in tumour-bearing mice, this change associates with higher GSSG levels, especially in advanced stages of cancer progression. Our results indicate that determination of glutathione status and oxidative stress-related parameters in blood may help to orientate cancer therapy in humans.     

Papaverine-induced changes in physiological characters of Ehrlich ascites tumor cell membranes

Physiological characters of Ehrlich ascites tumor cell membranes were altered by papaverine. The agent induced changes in membrane potential as monitored by cyanine dye (diS-C3-(5)) technique. Papaverine also strongly inhibited increase in membrane permeability to K+ ion induced by lysolecithin. In addition, papaverine inhibited oxygen uptake of the tumor cells and oxidative phosphorylation of their mitochondria, and slightly increased membrane fluidity. The results suggest that papaverine maintains compartmentation of K+ ion, energy metabolism, and membrane fluidity by regulating intracellular mitochondrial metabolism of Ehrlich ascites tumor cells.     

Enhancement of the efficiency of photodynamic therapy of tumours by t-butyl-4-hydroxyanisole

The effects of photodynamic therapy (PDT) alone and in combination with 3(2)-t-butyl-4-hydroxyanisole (BHA) on Ehrlich ascites carcinoma (EAC) cells have been investigated. BHA, a widely used food antioxidant, administered to the cells prior to light exposure is found to cause concentration-dependent alterations of the haematoporphyrin derivative (HpD)-based PDT. BHA (0.15 mM) causes a small (about 10%) inhibition in the rate of HpD-photosensitized injury of EAC cells. In contrast, upon increasing the concentration of BHA from 0.15 to 0.5 mM, a 1.3-fold enhancement in HpD-PDT efficiency is achieved. The cytotoxic effect on the cells treated with HpD-PDT and a higher concentration of BHA (0.5 mM) is additive. When BHA (0.5 mM) is given immediately after HpD-PDT, the combination is found to be three to four times more effective than when BHA is added to EAC cells before phototherapy. In this treatment regimen BHA acts synergistically with HpD-PDT. Such a difference in the action of BHA on the efficiency of HpD-PDT might be explained by the ability of BHA to inhibit the HpD-photosensitized destruction of some biomolecules. An enhancing action of BHA on the intensity of HpD-photosensitized death of tumour cells is also observed in vivo. Even a single dose of BHA (0.6 mM kg-1, 15 min after irradiation) causes (in an additive manner) an approximately two-fold increase in the efficiency of HpD-PDT of mice bearing Ehrlich ascites tumour (intraperitoneal transplantation). The results obtained indicate that the potentiating effect of BHA on the HpD-PDT could be caused by the impairment of the mitochondrial respiration, since there is a good correspondence between the concentration of BHA that increases the efficiency of PDT and the concentration that inhibits the oxygen consumption and dehydrogenase activity of EAC cells. The influence of BHA on the efficiency of PDT does not depend on the nature of the photosensitizer used; the effects with chlorin-e6 trimethyl ester are similar to that seen for HpD.     

Antitumour activity of mitoxantrone-loaded chitosan microspheres against Ehrlich ascites carcinoma

Glutaraldehyde cross-linked chitosan microspheres containing the antineoplastic agent mitoxantrone were prepared and the antitumour activity was evaluated against Ehrlich ascites carcinoma in mice by intraperitoneal injections. The tumour inhibitory effect was followed by monitoring animal survival time and change in body weight for a period of 60 days. While the mean survival time of animals which received 2 mg and 1 mg of free mitoxantrone intraperitoneally was 2.1 and 4.6 days, respectively, animals which received 2 mg mitoxantrone via microspheres showed a mean survival time of 50 days. Five out of 8 animals treated using microspheres lived beyond 60 days. The percentage ratio mean survival time of the treated group divided by the mean survival time of the untreated group for animals treated using mitoxantrone-loaded chitosan microspheres containing 2 mg of the drug was 290 compared with 12.2 for those which received 2 mg of the free drug. The antitumour effect of mitoxantrone-loaded microspheres against Ehrlich ascites carcinoma was much higher than that of doxorubicin-loaded microspheres reported by previous workers. Our data demonstrate the potential of mitoxantrone-loaded chitosan microspheres for sustained drug delivery to minimize drug toxicity and maximize therapeutic efficacy.     

Inhibitory effect of some esters of 4-hydroxy-5-nitro-4,5-dihydrothymine on the growth of tumor cells

New analogs of 4-hydroxy-5-nitro-4,5-dihydrothymine, namely 4-acyloxy-5-nitro-4,5-dihydrothymines (where: acyl = acetyl, benzoyl, mono- and dichloroacetyl) were synthesized and tested for their toxicologic and antitumor effects on Ehrlich ascites tumor cells and Ehrlich solid tumor. Best results were found for the mono- and dichloroacetyl derivatives.     

The nucleolar fibrillar centres in various cell types in vivo or in vitro

Nucleoli were studied in chick fibroblasts cultured in vitro, under normal or under experimental conditions, and in several mammalian cell types in vivo. All these cells frequently contain nucleoli with fibrillar centres. The nucleolar fibrillar centres are composed of fibrous material of low electron density and are always intimately associated with the dense fibrillar component. Their morphology is very similar to that analysed cytochemically in Ehrlich tumour cells. It therefore appears that they could be related to the nucleolar organizers as suggested in Ehrlich tumour cells.     

Crisis placement

The effect of the phosphatase inhibitor calyculin A (cal A) on the kinetic parameters of the Na+-coupled taurine uptake via the taurine transporter in the Ehrlich ascites tumour cells has been investigated. Preincubation with cal A (100 nM) reduces the initial taurine influx by about 20%, but has no effect on the diffusional component of the taurine influx or on the taurine release from cells suspended in isotonic or in hypotonic medium. Thus, cal A-sensitive phosphatases only affect taurine transport mediated by the Na+-dependent taurine transporter. Cal A increases the Michaelis-Menten constant for binding of taurine to the transporter from 31+/-6 to 45+/-4 microM and reduces the taurine transport capacity from 210+/-20 to 170+/-10 nmol x g dry wt(-1) x min(-1) [corrected]. The Michaelis-Menten constant for binding of Na+ to the taurine transporter is concomitantly increased from 96+/-11 to 129+/-8 mM and the Na+:taurine coupling ratio for activation of the transport cycle is reduced from 3.3+/-0.6 to 2.4+/-0.2. This suggests that cal A-sensitive phosphatases maintain a high affinity of the taurine transporter towards Na+ and taurine as well as a high taurine transport capacity in unpertubated Ehrlich cells.     

Regression of Ehrlich ascites carcinoma in BCG-sensitized mice

Intraperitoneal inoculation of CF1 mice with Bacillus Calmette-Guérin (BCG) protected many of them against the ascites form of Ehrlich carcinoma; and, for those that developed cancer, complete regression occurred in up to 50% of the cases at an advanced state of the neoplastic disease. In contrast, when a booster dose of BCG was administered in admixture with tumor cells, the incidence of the tumor was lower and tumor regressions were very rarely observed in mice that developed cancer. Trypan blue, an inhibitor of lysosomal enzymes of macrophages, was found to markedly suppress the natural (innate) antitumor resistance of control mice as well as the acquired resistance and tumor regressions of BCG-sensitized mice. Moreover, a comparison of the cytotoxic activity of the adherent (macrophages) and nonadherent (predominantly lymphocytes) cells isolated from the peritoneal cavity of BCG-sensitized mice, as measured by the inhibition of DNA synthesis, revealed that the effector cells were amongst the macrophages. In contrast, spleen macrophages were devoid of cytotoxicity. The spleen lymphocytes from both BCG-sensitized and control mice possessed about the same significant cytotoxic activity. These results indicate that the activated peritoneal macrophages, induced by a local injection of BCG, could play an important role in the antitumor immunity against Ehrlich carcinoma.     

Epithelioid leiomyosarcoma of the stomach with hemorrhagic ascites

In two men (69 and 65 years old) a gastric epithelioid leiomyosarcoma more than 25 cm in diameter first manifested itself by an increase in abdominal girth and ascites. In case 1, a cylindrical mass was palpable in the abdomen increasing in size over two years. Sonography and computed tomography showed a large cystic tumour which could not be related to any one organ. Cytology of the ascites fluid did not reveal any tumour cells. At operation a pedunculated tumour, 29 x 20 x 9 cm, was found, originating from the stomach. Six litres of ascites fluid were aspirated. There were no detectable abdominal metastases. The patient died nearly 4 years later after the tumour had been resected, three further laparotomies had to be performed because of extensive metastases to peritoneum and liver from the leiomyosarcoma. In case 2, no abdominal mass was palpable, but the patient's girth had increased over the last year. Sonography revealed a cystic tumour, about 30 cm in diameter. Examination of haemorrhagic ascites fluid (700 ml) did not show any tumour cells. The tumour, originating from the stomach wall, was excised (no metastases were seen). The patient remains symptom-free after 35 months.     

Persistence of colchicine resistance in Ehrlich-Lettré ascites tumors and cell strains

The effect of colchicine on mitoses of mutant HD33 Ehrlich-Lett?e ascites cells growing in vivo and in vitro was studied. HD33 mouse ascites tumors are colchicine-resistant. The LD50 of colchicine in mice bearing HD33 ascites tumors was 1.4 mg/kg body weight (b.w.), but a single dose of 3.33 mg colchicine/kg b.w. failed to suppress the anaphase of HD33 tumor mitoses for 24 h. No change in the level of colchicine resistance was observed after 269 weekly transplantations of HD33 ascites tumors without colchicine. In suspension culture, growth of HD33 ascites cells ceased at 1.5 x 10(-6) M colchicine. 10(-5) M colchicine suppressed the anaphase of HD33 mitoses and produced typical C-mitoses within one hour. The same effects on mitoses of colchicine sensitive Ehrlich ascites cells in vitro were achieved with 10(-6) M colchicine. In HD33 ascites cell cultures grown without colchicine, only a slight increase in colchicine sensitivity was registered after 5 years. Parallel cultures were propagated for the same period in the presence of 10(-7) M colchicine (HD33C ascites cells) without detectable growth alterations; the resistance level increased slightly. The limit of 10(-6) M colchicine was tolerated by the ascites cells in permanent culture without growth reduction (HD33CS ascites cells). 3H-colchicine binding studies suggest a permeability barrier of the plasma membrane as a mechanism of genetically fixed resistance.     

Purification and characterization of three acidic, cytotoxic phospholipases A2 from Indian cobra (Naja naja naja) venom

Three acidic phospholipases A2 (NN-I2c-PLA2, NN-I2d-PLA2 and NN-I2c-PLA2) were purified by successive chromatography of Indian cobra (Naja naja naja) venom on CM-Sephadex C-25, Sephadex G-50 and QAE Sephadex A-25 columns. They have molecular weights of 13,000-14,500 by sodium dodecyl sulphate polyacrylamide gel electrophoresis. They showed tryptophan specific fluorescence emission spectra (approximately 345 nm). All the three phospholipases A2 were enzymatically highly active with specific activities 9-17 micromol min(-1) mg(-1). They were non-lethal to mice when injected intraperitoneally in doses up to 10 mg kg(-1) body weight. They induced edema in mouse foot pads and were cytotoxic to Ehrlich ascites tumour cells. They did not exhibit direct haemolytic and anticoagulant activities.     

Cytocidal effect of alpha-methylornithine, a competitive inhibitor of ornithine decarboxylase, on ehrlich ascites tumor cells in vivo

The antiproliferative effect of alpha-methylornithine, an inhibitor of the initial and rate-limiting step in polyamine biosynthesis, was studied on Ehrlich ascites tumor cells grown in vivo. To assess drug-induced cell lethality, the loss of 125I from mice harboring 5-[125I] iodo-2'-deoxyuridine-labeled mouse tumor cells was determined during the course of alpha-methylornithine treatment. Mice that received the drug showed a significant increase in their rate of 125I excretion as compared to controls. This suggests that alpha-methylornithine kills an additional fraction of tumor cells beyond the fraction normally dying.     

An animal model of hepatotoxicity associated with halothane anesthesia

32P-Labeled phospholipids with specific activities up to 400 mCi/mmole as well as [32P]CDP-choline were prepared by cultivation of mouse fibroblasts or mouse Ehrlich ascites cells in the presence of [32P]orthophosphate. The method was also used to prepare [methyl-3H]choline-labeled glycerophospholipids from [3H]choline. The yields and the specific activities of the phospholipids were significantly lower when preparations of ox white blood cells were used.     

On the heterogeneity of native ribosomal subunits in Ehrlich-ascites-tumor cells cultured in vitro

Native small ribosomal subunits in cultured Ehrlich ascites tumor cells, analyzed by high-resolution CsCl isopycnic centrifugation, consist of at least five classes of particles. These particles with buoyant densities of 1.39, 1.42, 1.45, and 1.51 g/cm3 were designated as SI, SII, SIII, SIV and SV, respectively. They were different from the ribosome-derived 40-S subunits which have a density of 1.54 g/cm3. Native large ribosomal subunits consist of at least two classes of particles with densities of 1.57 g/cm3 (LI) and 1.59 g/cm3 (LII), respectively. The ribosome-derived 60-S subunits have the same buoyant density as LII particles. Labeling of Ehrlich ascites cells with radioactive uridine and a subsequent chase in the presence of RNA-synthesis inhibitors shows that radioactivity was incorporated first in precursor particles with a density of 1.48 g/cm3 and then subsequently appeared in SIII and SII particles. Met-tRNAf is found exclusively on native 40-S particles with densities of 1.42 and 1.49 g/cm3. This result has been observed in cells labeled with [35S] methionine in vivo as well as with tRNA charged in vitro. The possibility that SII particles contain 40-S initiation complexes is discussed.     

Reduced plaque accumulation on hydrocarbon thin film deposited on restorative acrylic polymers

The anti-tumour activity of acriflavine in combination with guanosine has been evaluated in solid or ascitic tumour-implanted animal models. Guanosine is known to potentiate the anti-tumour effects of some chemotherapeutic agents. Administration of acriflavine (15 mg kg-1 day-1, i.m., 14 days) to ICR mice subcutaneously implanted with Ehrlich carcinoma resulted in approximately 30% inhibition in tumour growth. In contrast, minor tumour growth inhibition was observed in animals treated with guanosine at the same daily dose. Treatment of animals with both acriflavine and guanosine (AG60, 1:1, w/w) at 30 mg kg-1 resulted in approximately 65% inhibition in tumour growth rate. Whereas treatment with acriflavine or guanosine resulted in 70% or 30% decrease in tumour weight, respectively, treatment of tumour-implanted mice with AG60 (30 mg kg-1) resulted in a 96% decrease in tumour weight, relative to control, 14 days after tumour-cell implantation. Dose-related inhibition in tumour growth rate was also observed in animals treated with AG60, with maximum (65%) inhibition noted at a dose of 30 mg kg-1 (ED50 23 mg kg-1). Suppression of body weight increase and elevated plasma glucose levels by acriflavine or AG60 indicated that glucose utilization might be impaired. The anti-tumour effect of AG60 was also determined in CDF1 mice intraperitoneally implanted with Ehrlich ascitic tumour. Ehrlich ascitic tumour proliferation was completely suppressed by AG60 (30 mg kg-1, i.p.). Microscopic analyses of intraperitoneal touch-prints revealed that AG60 was more effective in suppressing tumour proliferation than acriflavine alone. Fluorescent microscopic examinations demonstrated that acriflavine avidly bound with Yac-1 cell plasma membrane, leading to morphological changes in the cells, such as bleb formations, swelling and ballooning. The time-related changes in tumour cell morphology by acriflavine or AG60 might represent energy depletion, followed by osmotic lysis as a result of cationic influx. Enhanced anti-tumour activity of acriflavine in combination with guanosine might be explained by the blocking of nutrient transport through selective acriflavine binding with plasma membrane and by concomitant guanosine perturbation of cellular ATP production. This study demonstrates that guanosine enhances the anti-tumour effects of acriflavine against a variety of cancer cells without serious adverse effects, providing a preclinical basis for potential application of this combination against cancer proliferation.