Aberrant 3H labelling of ATP during in vivo labelling of Ehrlich mouse ascites tumour cells with [2-3H]inositol is significant in the study of isomers of InsP3 and InsP4.
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ABSTRACT:
SUBMITTER: Christensen S
PROVIDER: S-EPMC1138244 | biostudies-other | 1994 Jun
REPOSITORIES: biostudies-other
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Project description:Cardiomyopathy is one of the characteristic features of cancer. In this study, we establish a suitable model to study breast cancer-induced cardiomyopathy in mice. We used Ehrlich Ascites Carcinoma cells to induce subcutaneous tumor in 129/SvJ mice and studied its effect on heart function. In Ehrlich Ascites Carcinoma bearing mice, we found significant reduction in left ventricle wall thickness, ejection fraction, and fractional shortening increase in left ventricle internal diameter. We found higher muscle atrophy, degeneration, fibrosis, expression of cell-adhesion molecules and cell death in tumor-bearing mice hearts. As observed in cancer patients, we found that mTOR, a key signalling molecule responsible for maintaining cell growth and autophagy was suppressed in this model. Tumor bearing mice hearts show increased expression and nuclear localization of TFEB and FoxO3a transcription factors, which are involved in the upregulation of muscle atrophy genes, lysosomal biogenesis genes and autophagy genes. We propose that Ehrlich Ascites Carcinoma induced tumor can be used as a model to identify potential therapeutic targets for the treatment of heart failure in patients suffering from cancer-induced cardiomyopathy. This model can also be used to test the adverse consequences of cancer chemotherapy in heart.
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Project description:The InsP3R Ca2+ release channel has a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca2+]i. To determine if relieving Ca2+ inhibition is sufficient for channel activation, we examined single-channel activities in low [Ca2+]i in the absence of InsP3, by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent channel activities with low open probability Po ( approximately 0.03) were observed in [Ca2+]i < 5 nM with the same frequency as in the presence of InsP3, whereas no activities were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i of the channel to be 1.2-4.0 nM in the absence of InsP3, and demonstrate that the channel can be active when all of its ligand-binding sites (including InsP3) are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies of [Ca2+]i and InsP3 regulation of steady-state channel gating behavior of types 1 and 3 InsP3R isoforms, including spontaneous InsP3-independent channel activities, the tetrameric channel can adopt six different conformations, the equilibria among which are controlled by two inhibitory and one activating Ca2+-binding and one InsP3-binding sites in a manner outlined in the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the Ca2+ affinities of the high-affinity inhibitory sites in different conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent low-affinity inhibitory site. The model also suggests that besides the ligand-regulated gating mechanism, the channel has a ligand-independent gating mechanism responsible for maximum channel Po being less than unity. The validity of this model was established by its successful quantitative prediction of channel behavior after it had been exposed to ultra-low bath [Ca2+].
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Project description:A novel method to separate [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 in tissue extracts is described. It is based on the selective metabolism of Ins(1,3,4)P3 by a crude cerebral supernatant in a Mg2+-free buffer followed by separation of [3H]inositol trisphosphates using conventional anion-exchange chromatography. Evaluation of the assay was performed using [3H]Ins(1,3,4)P3 standards and tissue extracts containing different proportions of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3. Parallel h.p.l.c. separations of extracts established the selective and complete metabolism of [3H]Ins(1,3,4)P3 under the above conditions and demonstrated that the enzymic method provides an accurate estimate of the trisphosphate isomers in rat cerebral cortex, parotid gland and bovine tracheal smooth muscle.
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Project description:The aim of this study is to investigate the antitumor activity and possible molecular mechanism of Phenethyl isothiocyanate (PEITC) against Ehrlich ascites carcinoma in-vivo and in-vitro. In-vivo, ascetic fluid volume, body weight, serum malondialdehyde (MDA) level and total antioxidant capacity (TAC) were determined using Ehrlich ascites carcinoma (EAC) bearing mice. In-vitro, MTT assay was used. RT-PCR was used to investigate role of PEITC in apoptosis by analyzing the expression of Bax, caspase-9, and Bcl-2 genes. The effect of PEITC on caspase-9 enzyme activity was also tested. PEITC and/or Doxorubicin (Dox) treatment significantly suppressed EAC growth as compared to EAC/oil control mice. PEITC treatment showed a dose-dependent inhibition of EAC cells as indicated by MTT assay. We found that significant increase in MDA level and decrease in TAC caused by Dox treatment were significantly reduced by combination with PEITC treatment. Bax, caspase-9 genes' expression and caspase-9 enzymatic activity were significantly increased, while Bcl-2 gene expression was significantly decreased in PEITC treated mice. PEITC may act as a promising anticancer agent either alone or more effectively in combination with Dox through apoptotic cell death induction.
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Project description:Cancer cells exhibit an increased glycolysis rate for ATP generation (the Warburg effect) to sustain an increased proliferation rate. In tumor cells, the oxidation of pyruvate in the Krebs cycle is substituted by lactate production, catalyzed by LDH. In this study, we use ethoxyquin (EQ) as a novel inhibitor to target LDH in murine Ehrlich ascites carcinoma (EAC) and as a combination therapy to improve the therapeutic efficacy of the conventional chemotherapy drug, cisplatin (CIS). We investigated the anti-tumor effect of EQ on EAC-bearing mice and checked whether EQ can sustain the anti-tumor potential of CIS and whether it influences LDH activity. Treatment with EQ had evident anti-tumor effects on EAC as revealed by the remarkable decrease in the expression of the anti-apoptotic gene Bcl-2 and by a significant increase in the expression of apoptotic genes (BAX and caspase-3). EQ also caused a significant decrease in the autophagic activity of EAC cells, as shown by a reduction in the fluorescence intensity of the autophagosome marker. Additionally, EQ restored the altered hematological and biochemical parameters and improved the disrupted hepatic tissues of EAC-bearing mice. Co-administration of EQ and CIS showed the highest anti-tumor effect against EAC. Collectively, our findings propose EQ as a novel inhibitor of LDH in cancer cells and as a combinatory drug to increase the efficacy of cisplatin. Further studies are required to validate this therapeutic strategy in different cancer models and preclinical trials.
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Project description:Ehrlich ascites-tumour cells were investigated with regard to their stability to transport L-lactate by measuring either the distribution of [14C]lactate or concomitant H+ ion movements. The movement of lactate was dependent on the pH difference across the cell membrane and was electroneutral, as evidenced by an observed 1:1 antiport for OH- ions or 1:1 symport with H+ ions. 2. Kinetic experiments showed that lactate transport was saturable, with an apparent Km of approx. 4.68 mM and a Vmax. as high as 680 nmol/min per mg of protein at pH 6.2 and 37 degrees C. 3. Lactate transport exhibited a high temperature dependence (activation energy = 139 kJ/mol). 4. Lactate transport was inhibited competitively by (a) a variety of other substituted monocarboxylic acids (e.g. pyruvate, Ki = 6.3 mM), which were themselves transported, (b) the non-transportable analogues alpha-cyano-4-hydroxycinnamate (Ki = 0.5 mM), alpha-cyano-3-hydroxycinnamate (Ki = 2mM) and DL-p-hydroxyphenyl-lactate (Ki = 3.6 mM) and (c) the thiol-group reagent mersalyl (Ki = 125 muM). 5. Transport of simple monocarboxylic acids, including acetate and propionate, was insensitive to these inhibitors; they presumably cross the membrane by means of a different mechanism. 6. Experiments using saturating amounts of mersalyl as an "inhibitor stop" allowed measurements of the initial rates of net influx and of net efflux of [14C]lactate. Influx and efflux of lactate were judged to be symmetrical reactions in that they exhibited similar concentration dependence. 7. It is concluded that lactate transport in Ehrlich ascites-tumour cells is mediated by a carrier capable of transporting a number of other substituted monocarboxylic acids, but not unsubstituted short-chain aliphatic acids.
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Project description:A simple method has been developed for the preparation of 3H-labelled bilirubin IX-alpha from bilirubin dimethyl ester. The label is located in the propionic acid side chains, and there is no isomerization of the bilirubin during the preparation.
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