Capacity for differentiation and normalization of populations of tumor cells transplanted into the anterior chamber of the eye. I. Changes in the pleomorphic rhabdomyosarcomas MKh-III and A-7 of mice

Tumors transplanted into the eye anterior chamber (EAC) were atypic, more differentiated morphologically and with higher activity of H-forms of LDH, compared to the same tumors grown in the subcutaneous connective tissue (SCT). Besides, in the EAC, the tumors were less karyotypically heterogeneous and had lesser mitotic indices, than in the SCT. After cultivation in the EAC, populations of tumor rhabdomyoblasts displayed a decreased transplantability in SCT, i.e. the usual site of their implantation. These results may suggest that the proliferation of tumor cell populations in the EAC, i.e. in the immunologically privileged place, can be accompanied by the reducing of their malignancy up to the total loss of the common tumorigenic ability. Even the cell populations of tumor rhabdomyoblasts from the late stages of tumor progression can undergo such a process of normalization.     

Capacity for differentiation and normalization of tumor cell populations in transplantation into the anterior chamber of the eye. III. Rhabdomyosarcoma A-7 clones

Four clone lines of transplantable cell polymorphic rhabdomyosarcoma A-7 were investigated during transplantation to the subcutaneous connective tissue (SCT) and into eye anterior chamber (EAC). Cell morphology of transplants was studied by light and electron microscopy, the activity of their LDH M- and H-subunits was examined cytochemically, and the quantity of their nuclear DNA--cytophotometrically. In the case of A-7/1, A-7/2 and A-7/3 cell lines of EAC transplants we noticed a decrease in cell element kataplasia levels, differences in LDH M- and H-form ratio, reduction in the karyotype variability. Transplants of A-7/4 clone line were similar in SCT and EAC for all the signs studied. The results obtained show that the transplantable cell polymorphic rhabdomyosarcoma A-7 is heterogeneous for its differentiation and normalizing capacities during EAC proliferation. The data reported elsewhere concerning capability of four lines of murine rhabdomyosarcomas to normalize in EAC are discussed, and some possible mechanisms of this effect are regarded.     

Lactate dehydrogenase-elevating agent is responsible for interferon induction and enhancement of natural killer cell activity by inoculation of Ehrlich ascites carcinoma cells into mice

Inoculation of Ehrlich ascites carcinoma cells (EAC) into the peritoneal cavities of outbred ddY mice induced interferon (IFN) in the circulation. The maximum titer (1,280 U) was obtained at 24 hr after inoculation. This induced IFN had the characteristics of type I IFN, i.e., stability at pH2 and lability at 56 C. An increase in natural killer cell (NK) activity was also observed for the first 3 days after inoculation. In addition, plasma lactate dehydrogenase (LDH) activity was elevated in these mice. Inoculation of ascitic fluid or serum of EAC-bearing mice into normal mice increased plasma LDH activity six- to sevenfold over normal levels and elevated activities persisted throughout the life of the mice. These results suggest that the LDH-elevating agent was responsible for IFN induction and for enhancing NK activity. Because lactate dehydrogenase-elevating virus (LDV) can be eliminated from tumor cells by passage in vitro, we attempted to grow EAC in tissue culture for several months and re-examined whether the inoculation of such cells could elevate plasma LDH activity induce IFN and enhance NK activity. The results showed that inoculation of the passaged cells had no effect on these activities in normal mice. Therefore, we concluded that the IFN inducer was LDV which contaminated the EAC and then enhanced the NK activity. N-tropic murine leukemia virus also contaminated EAC, but this virus was not responsible because cultured cells of EAC still shed this virus.     

Dual inhibition of glycolysis and autophagy as a therapeutic strategy in the treatment of Ehrlich ascites carcinoma

Cancer cells have extra biosynthetic demands to sustain cell growth and redox homeostasis. Glycolysis and autophagy are crucial to fuel and recycle these biosynthetic demands. This plasticity of cancer cell metabolism participates in therapy resistances. The current study was designed to assess the therapeutic efficacy of dual targeting of glycolysis and autophagy in cancer. Using 3‐bromopyruvate (3‐BP; antiglycolytic inhibitor) and hydroxychloroquine (HCQ; autophagy inhibitor), we demonstrate their antitumor activity in Ehrlich ascites carcinoma (EAC)‐bearing mice. A combination of 3‐BP and HCQ significantly decreases tumor ascitic volume and cell count as compared with the EAC group and individual treatment groups. The enhanced antitumor activity is accompanied by hexokinase inactivation, inhibition of cellular protective autophagy, elevated antioxidant activity, and reduced oxidative stress levels. Together, these results suggest targeting both pathways in cancer as an effective therapeutic strategy. Further studies are required to validate this strategy in different cancer models and preclinical trials.     

Exogenous DNA transcription in cells with their native DNA inhibited. 4. Demonstration of specific antigens codified by exogenous DNA

It is possible to obtain antisera to cancer-specific antigens of mouse Ehrlich ascites cancer (EAC) cells when chimera rabbits previously made unresponsive to immunological stimulation by normal mouse cells antigens are immunized with mouse cancer cells. Employing this specifically anti-cancerous immunoserum this work shows that EAC cells and TC-SV40 cells contain cross-reacting cancer-specific antigens. Exogenous DNA from EAC cells is demonstrated as being able to display its coded information when it is incorporated to endogenous TC-SV40 cell DNA whose activity has been previously inhibited by 5'-bromodeoxyuridine.     

Isolation of fatty acids from the mycelium ofPenicillium stipitatum thom and their effect on ehrlich ascites carcinoma cells

A total of 5 fractions were isolated from the mycelium ofPenicillium stipitatum Thom obtained by submerged cultivation. Three of them inhibited the incorporation of¹⁴C-labelled precursors into Ehrlich ascitic cells (EAC) at concentrations lower than 100 μg/ml. The fraction M-72-3, inhibiting mainly adenine incorporation, was further separated into 6 fractions. The highest effect on EAC cells was exerted by subfraction IV which consisted of free fatty acids; its main effective components were oleic linoleic and linolenic acid. Their cyto-inhibitory effect on EAC cells was confirmed by their application in a pure form.     

Cell populations of transplantable murine tumors of various degrees of histogenesis during their proliferation in the anterior chamber

Six transplantable murine tumors of different histogenesis were investigated after transplantation to subcutaneous connective tissue (SCT) and the eye anterior chamber (EAC). Cell morphology was studied using light microscopy. DNA contents in the nuclei of tumor cells were investigated with flow cytometry technique. LDH isoenzymes were studied using electrophoresis in polyacrylamide gel. In the case of tumors with near diploid modal class, a redistribution of LDH isoenzyme activity and an increase in morphological differentiation level were obtained. In the case of tumors with modal class differing from the diploid one, a morphological structure changes were revealed, but there were no differences in LDH isoenzyme activity. The data obtained show that the capability of increasing morphological and biochemical differentiation level after cultivation in the EAC of murine transplantable tumors remains even on the late stages of progression in tumors of different histogenesis with near diploid value of modal class.     

Initiation of DNA synthesis in the liver and other tissues of adult mice by a growth factor (EACF) isolated from acellular fluid of the Ehrlich ascites carcinoma

The mitogenic effect of a new growth factor that we recently isolated from the acellular ascitic fluid of the Ehrlich ascites carcinoma grown in vivo was examined. We have called this factor EACF (Ehrlich ascites carcinoma factor). EACF caused initiation of DNA synthesis in the liver, submandibular gland, exorbital lacrimal gland and epithelium of the tongue of adult mice after i.p. injection at a protein concentration of 3 micrograms per 25 g of body weight. For all tissues examined, except the tongue, EACF initiated DNA synthesis at about 48 to 60 h after injection, with the maximum effect at approx. 85 h, and the stimulatory effect lasting approx. 60 h. The initiation of DNA synthesis in liver, which is normally characterized by only an occasional cell passing through the S phase, by EACF is of particular interest. The initiation of DNA synthesis in the liver was not prevented by hypophysectomy. Evidence also indicates that a similar heat-labile growth promoting factor(s) is present in calf serum.     

Antitumor activity of equinatoxin.

Equinatoxin, a highly basic protein extracted from Actinia equina, causes an increase in the survival time of mice bearing the ascitic form of Ehrlich carcinoma, whereas it has no effect on L1210 leukaemia. When tested for in vitro cytotoxicity by the dye exclusion test, it shows a potent activity on both tumour cell lines, with ED50 of a few ng/ml. Higher concentrations produce an extensive lysis of the cells. The cytotoxic effects of Equinatoxin are inhibited by phospholipids, thus suggesting that its mechanism of action may be related to interactions with lipids or other charged components of cell membrane. The observed lack of in vivo activity against L1210 leukaemia presumably is due to poor systemic absorption of the protein and/or neutralization by serum factors.     

Iron N-(2-hydroxy acetophenone) glycinate (FeNG), a non-toxic glutathione depletor circumvents doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo

Multidrug resistance-associated protein 1 (MRP1) reduces intracellular anticancer drug accumulation either by co transporting them with glutathione (GSH) or extruding drug-GSH conjugates outside of the cell. Thus, MRP1 confers multidrug resistance (MDR) and worsen successful chemotherapeutic treatment against cancer. Although the exact mechanism of MRP1 involved in MDR remains unknown, the elevated level of intracellular GSH is considered as a key factor responsible for MDR in cancer. Hence the quest for non-toxic molecules that are able to deplete intracellular GSH has profound importance to subdue MDR. The present preclinical study depicts the resistance reversal potentiality of an iron complex; viz. Ferrous N-(2-hydroxy acetophenone) glycinate (FeNG) developed by us in doxorubicin resistant Ehrlich ascites carcinoma (EAC/Dox) cells. FeNG potentiate cytotoxic effect of doxorubicin on EAC/Dox cells ex vivo and also increases the survivability EAC/Dox bearing Swiss albino mice in vivo as well. Moreover, in vivo administration of FeNG significantly depletes intracellular GSH with ensuant increase in doxorubicin concentration in EAC/Dox cells without alternation of MRP1 expression. In addition, intra-peritoneal (i.p.) application of FeNG in normal or EAC/Dox bearing mice does not cause any systemic toxicity in preliminary trials in mouse Ehrlich ascites carcinoma model. Therefore, the present report provides evidence that FeNG may be a promising new resistance modifying agent against drug resistant cancers.     

Bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex a new apoptotic agent through Flk-1 down regulation,caspase-3 activation and oligonucleosomes DNA fragmentation

New bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex was synthesized and characterized. In vivo anti-angiogenic activities of bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex against Ehrlich ascites carcinoma (EAC) cells are described. The newly synthesized complex resulted in inhibition of proliferation of EAC cells and ascites formation. The anti-tumor effect was found to be through anti-angiogenic activity as evident by the reduction of microvessel density in EAC solid tumors. The anti-angiogenic effect is mediated through down-regulation of VEGF receptor type-2 (Flk-1). The complex was also found to significantly increase the level of caspase-3 in laboratory animals compared to the acridine ligand and to the control group. This was also consistent with the DNA fragmentation detected by capillary electrophoresis that proved the apoptotic effect of the new complex. Our complex exhibited anti-angiogenic and apoptotic activity in vivo, a thing that makes it a potential effective chemotherapeutic agent. The interaction of calf thymus DNA (ct-DNA) with bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex has been investigated using fluorescence technique. A competitive experiment of the europium(III)-acridine complex with ethidium bromide (EB) to bind DNA revealed that interaction between the europium(III)-acridine and DNA was via intercalation. The interaction of the synthesized complex with tyrosine kinases was also studied using molecular docking simulation to further substantiate its mode of action.     

Bioactive component,cantharidin from <Emphasis Type="Italic">Mylabris cichorii</Emphasis> and its antitumor activity against Ehrlich ascites carcinoma

The anticancer activity of the extract of blister beetle, Mylabris cichorii has been documented earlier by us. In the present study, the active principle of M. cichorii was isolated and its anticancer efficacy was evaluated against murine Ehrlich ascites carcinoma (EAC). The isolated bioactive compound was characterized to be cantharidin which showed potent antitumor activity and inhibited the proliferation of Ehrlich ascites carcinoma, both in vivo and in vitro. Cantharidin-treated EAC-bearing mice showed about 82% increase in lifespan at the dose of 0.5 mg/kg/day. In vitro cytotoxicity assay with the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test revealed about 50% cell death at the concentration of 25.8 μg/ml. The fluorescence and transmission electron microscopy revealed that EAC cells treated with cantharidin depicted typical apoptotic morphology with chromatin condensation, nuclear fragmentation into discrete masses, and plasma membrane blebbing which deduce towards the death of these cells. Histological examination of the kidney of cantharidin-treated mice showed glomerular and tubular congestion with abnormal Bowman’s capsule, thus, indicating a renal toxicity in the host. Cantharidin-induced renal damage in the host was also manifested by the decreased lactate dehydrogenase isozymes and its possible release from the cells.     

Long-Term Caffeine Inhibits Ehrlich Ascites Carcinoma Cell-Induced Induction of Central GABAergic Activity

Long-term administration (for 22–27 consecutive days) of caffeine (20 mg/kg/day p.o) developed tolerance to this drug by upregulating the central GABAergic activity. Development of Ehrlich ascites carcinoma (EAC) cell induced the whole brain GABAergic activity. But pre-treatment of caffeine and continuation of its treatment in the course of development of EAC cells restored the EAC cell-induced change of GABAergic activity to control values. Thus, it may be concluded that caffeine (adenosine receptor antagonist) suppresses the EAC cell-induced induction of whole brain GABAergic activity in mice.     

Antitumor potential of Castanopsis indica (Roxb. ex Lindl.) A. DC. leaf extract against Ehrlich's ascites carcinoma cell

Methanol extract of C. indica (MECI) leaves showed direct cytotoxicity on Ehrlich ascites carcinoma (EAC) cell in a dose dependant manner and there was significant decrease in the tumor volume, viable cell count, tumor weight and elevated the life span of EAC tumor bearing mice. Hematological profile and biochemical estimations were significantly restored to normal levels in MECI treated as compared to EAC control mice. MECI treatment significantly modulated the tissue antioxidant assay parameters as compared to the EAC control mice. The results revealed that MECI possesses significant dose dependent antitumor potential which may be due to its cytotoxicity and antioxidant properties.     

The effect of ascitic fluid on the growth of Ehrlich tumor and Lewis carcinoma]

In the study of the effect of ascitic fluid and dialysate of Ehrlich ascites tumor cells (m.m. less than 15 kDa) on the growth of Ehrlich and Lewis carcinoma it was found that the ascitic fluid significantly decreased the size of Ehrlich tumor (by more than 50% on day 9-25 after the tumor cell inoculation). It also reduced Lewis carcinoma tumor volume by more than 30% during 3 weeks after the tumor cells inoculation. Dialysate of Ehrlich tumor cells significantly inhibited the growth of Ehrlich tumor too. It is suggested that this test-system simulates inhibition of a small tumor by a big tumor in vivo.     

Effect of audiogenic convulsions on the activity of n- and m-forms of lactate dehydrogenase in the neurons and neuroglia of different sections of central nervous system]

It was shown spectrophotometrically that in Krushinsky-Molodkina and Wistar rats the ratio of the activity of the aerobic H-forms of lactic dehydrogenase (LDH) to the activity of the anaerobic M-forms was higher in the neurons of the cerebral cortex and the Purkinje's cells of the cerebellum and in their glial cells-satellites than in the motor neurons of the anterior horns of the spinal cord and their perineuronal glia. In Krushinsky-Molodkina rats (with genetically-determined high sensitivity to audiogenic convulsions) epileptiform attacks under the effect of sound were accompanied by a marked activation of both the H- and the M-forms of LDH in the cortical neurons in the absence of any shifts in the perineuronal glia. On the contrary, the activity of all the forms of LDH was unchanged in the spinal motor neurons, whereas in the neuroglia cells surrounding these neurons there was a distinct increase in the activity of the H-forms of LDH. In the Purkinje's cells of the cerebellum an increase and in the glial cells- satellites -- a reduction of the activity of the M-forms of LDH in case of convulsions was seen.     

Synthesis and antiproliferative activity of some novel derivatives of diospyrin, a plant-derived naphthoquinonoid

Derivatisation of diospyrin, a bisnaphthoquinonoid isolated from Diospyros montana Roxb., led to the modification of its inhibitory activity, in vitro, towards a murine tumour model, Ehrlich ascites carcinoma (EAC), and two human cancer cell lines, viz., malignant skin melanoma (A375) and epidermoid laryngeal carcinoma (Hep2). Among the novel derivatives, an epoxide exhibited the maximum antiproliferative activity (IC(50) values in the range of 0.03-0.21 microM) and a comparatively lower toxicity (IC(50) approximately 98 microM) in normal human peripheral blood mononuclear cells (PBMC). This compound might provide a novel 'lead' for the development of clinically effective antiproliferative agents against cancer.     

Glucose decreases respiratory control ratio in EL-4 tumor cells.

EL-4 ascites thymoma cells are shown to have high aerobic glycolysis and decreased Pasteur effect. At the same time, glucose produces a much smaller inhibitory effect on cell respiration (Crabtree effect) than in Ehrlich ascites carcinoma (EAC) cells. In intact EL-4 cells, the respiratory control ratio (RCR) was found to be 6.2 with endogenous substrates and 8.0 with glutamine. Glucose decreased the RCR to 3.2, by stimulating the state 4 respiration. In rat thymocytes and EAC cells, such an effect of glucose was absent (RCR of 7.0 and 7.2, respectively). It is suggested that in EL-4 tumor cells, the high aerobic glycolysis and small Crabtree effect may be due to glucose-induced 'uncoupling' of oxidation and phosphorylation.     

Glyceraldehyde-3-phosphate dehydrogenase from Ehrlich ascites carcinoma cells its possible role in the high glycolysis of malignant cells.

Glyceraldehyde-3-phosphate dehydrogenase has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells. The enzyme is quite active over a pH range of 7.5-9.0 with an optimum pH of 8.4-8.7. The specific activity of the enzyme is much higher than that from other normal sources. In contrast to enzyme obtained from rabbit muscle, the EAC cell enzyme is not significantly inhibited by physiological concentrations of ATP at physiological pH. Kinetic studies using different substrates and inhibitors indicate that the properties of the EAC cell enzyme are significantly different from those of glyceraldehyde-3-phosphate dehydrogenase obtained from other normal sources. The striking dissimilarity of the malignant cell glyceraldehyde-3-phosphate dehydrogenase compared with this enzyme from other normal sources, particularly in respect to the interaction with ATP, may in part explain the high glycolysis of malignant cells.     

Carcinoma infection and immune systems of Ehrlich ascites carcinoma-bearing mice treated with structurally similar sulfonium compounds

The effects of intraperitoneal administration of dimethylsulfonioacetate (DMSA), dimethlsulfoniopropionate (DMSP), and methylmethionine (MeMet) solutions (10 mM each) on the body weights and the hematological parameters (red and white blood cells) of Ehrlich ascites carcinoma (EAC)-bearing mice were examined for up to 10 d. Body weights significantly increased in the EAC-bearing mice treated with and without MeMet in contrast to those with DMSA and DMSP. This increase was attributed to the increased amounts of ascitic fluid. EAC-bearing mice with and without MeMet both showed abnormal values of hematological parameters, while those with DMSA and DMSP exhibited almost normal levels on the 10th day.