Effects of Green cardamom (Elettaria cardamomum Maton) and its combination with cyclophosphamide on Ehrlich solid tumors.

BACKGROUND: Cardamom (Elettaria cardamomum) is a spice and exhibits potent antioxidant and biological activities through distinct molecular mechanisms. However, the anticancer effect of cardamom was not explored yet in Ehrlich solid tumor (EST)-bearing mice. OBJECTIVES: This investigation was aimed to evaluate the anti-cancer effects of green cardamom (GCar) alone or combined with the anti-cancer drug cyclophosphamide in an in vivo model to explore its mechanistic role in tumor cell death in EST-bearing mice. METHODS: Ehrlich ascites tumor cells were injected in the mice and 5 days later the animals treated with GCar and/or cyclophosphamide for 10 days. Twenty-four hours from the last treatment, animals were sacrificed for the different measurements. RESULTS: Data recorded for tumor size, percentage of tumor growth inhibition, tumor growth delay and mean survival time of EST-bearing mice demonstrated the effective role of GCar alone or combined with CPO as a promising anti-cancer agent because it reduced tumor size. GCar elevated the mean survival time of EST-bearing mice compared to that of untreated EST and EST + CPO groups. Analysis of qPCR mRNA gene and protein expression revealed that GCar alone or combined with CPO were promising anticancer agents. After the treatment of EST with GCar, the apoptotic-related genes and proteins were significantly modulated. GCar induced markedly significant decreases in oxidative stress biomarkers and a significant increment in glutathione levels and that of antioxidant enzymes. With a marked diminish in liver and kidney function biomarkers. CONCLUSION: The results revealed that GCar could serve as an apoptotic stimulator agent, presenting a novel and potentially curative approach for cancer treatment, inducing fewer side effects than those of the commercially used anti-cancer drugs, such as CPO.

[Kinetic features of ascites and solid Ehrlich tumors].

Such biological parameters as tumor volume, Ki-67 and p53, which characterize the development of ascites and solid tumor of Ehrlich were evaluated. The kinetic curve of growth of ascites tumor was S-shaped (Gomperts) while that of the solid one--cubic (Speer-Retsky). Ki-67 expression level was found to be in cyclic correlation with duration (3 and 6 day intervals) which might be worth considering when working out therapeutic procedure. Moreover, no increase in cell death was observed when tumor growth slowed down.

The role of copper in development of drug resistance in murine carcinoma.

Multidrug resistance (MDR) is a major obstacle to successful application of cancer chemotherapy and also a basic problem in cancer biology. Studies on the molecular basis of MDR have revealed that a number of proteins over express in multidrug resistant cells viz., multidrug resistant MDR1 gene product P-glycoprotein, the multidrug resistance-associated protein (MRP) and enzymes associated with the glutathione (GSH) metabolism. Decreased expression or altered activity of topoisomerase II has also been implicated in MDR. In the present investigation a number of changes in phase II detoxification parameters have been noticed in drug resistant cells but the novel aspect of the present report is the observation that the metal copper is involved in drug resistance. Although copper plays important roles in many human and other biological systems and even in the treatment of cancer but the relation of Cu and drug resistance has not so far been studied in detailed. The present report describes the novel findings that the level of copper increases with the development of drug resistance in Ehrlich ascites carcinoma and in Lewis lung carcinoma cells and also in serum of mice bearing drug resistant cancer cells compared to mice bearing drug sensitive cells; the work indicates the important aspect of treating drug resistant cancer patients by lowering Cu level in the cancerous cells and serum prior to treatment.

[The effect of phorbol ester in a wide range of concentrations on the lipid structure of microsome membranes of tumor cells in vitro]. / Deistvie forbolovogo éfira v shirokom diapazone kontsentratsii na strukturu lipidov mikrosomal'nykh membran opukholevykh kletok in vitro.

The action of 12-O-tetradecanoyl-13-acetate (TPA) in vitro in a wide range of concentration from 10(-3) mol/l down to ultra-low doses 10(-23) mol/l and dilution 10(-24) mol/l on the microsome membranes isolated from tumor--Ehrlich ascite carcinoma (EAC) has been studied by ESR-method using two spin probes: 5- and 16-doxyl stearates (5- and 16-DS) localized in the different regions of lipid bilayer. From the ESR spectra obtained it was calculated the following parameters: an order of the long axis 5-DS (S) related to order of the fatty acids chains in the lipid bilayer; two rotation correlation times (Tc1 and Tc2) of 16-DC to estimate a microviscosity value and structural-sensitive ones. It was found the stage changes of all these parameters (increase and decrease) as compared with control level (the membranes untreated by TPA) depending on TPA concentration into the range of 10(-3)-10(-24) mol/l; in particular, the most significant shape changes of structural-sensitive parameters have been observed at TPA doses below 10(-16) mol/l. It is concluded that tumor membranes are very sensitive to TPA action in vitro in a wide range of concentration included ultra-low doses.

The pump and leak steady-state concept with a variety of regulated leak pathways.

The paper will reflect on how Ussing has affected my own scientific work and how he created much of the framework within which I have been working. I have used five examples: (i) The first description of a 1:1 exchange diffusion was introduced by Ussing in 1947 and has been found to be of great physiological significance in most cells. We found that Cl- transport in Ehrlich ascites tumor cells (EATC) was completely dominated by an exchange diffusion process, as defined by Ussing, and, thus, the Cl- conductance was much lower than previously estimated from measurements of unidirectional tracer fluxes. This had a major influence on my later description of a swelling-activated Cl- conductance. (ii) The pump-leak steady-state concept for cell volume control was introduced by Krogh in 1946, but it was developed in detail by Leaf and Ussing in 1959. This concept was the basis for me and others, when we later found that the passive ion leaks play an active role in cell volume control. (iii) The use of isotopes and Ussing's famous flux ratio equation provided an ingenious instrument for distinguishing the various transport routes. We used this to identify the Na,K,2Cl cotransport system as accounting for maintaining a [Cl-]i in the EATC far above thermodynamic equilibrium, as well as accounting for the ion uptake during a regulatory volume increase (RVI) in EATC, similar to what Ussing had found in frog skin. (iv) Short-circuit current setup in the Ussing chamber is still used in laboratories around the world to study ion transport across epithelia. A few results on Cl- transport across the operculum epithelium of the small eurohaline fish Fundulus heteroclitus mounted in small Ussing chambers are presented. (v) Shrinkage-activated Na+ conductance and its possible role in isotonic secretion in frog skin glands is finally discussed.

A fraction isolated from Ehrlich ascites carcinoma as an antitumor and differentiating agent against human leukemic cell ML-2.

Lipopolysaccharide fraction isolated from Ehrlich ascites carcinoma (E-LPS) was investigated as an antitumor agent against human leukemia cell ML-2. Marked cell growth inhibition was observed with ML-2 cell accompanied by inhibition of DNA synthesis and perturbation of cell cycle. Induction of differentiation in treated ML-2 cells was observed as indicated by morphological maturation, NBT reducing activity and indirect immunofluorescence.

Characterisation of a cell swelling-activated K+-selective conductance of ehrlich mouse ascites tumour cells.

The K+ and Cl- currents activated by hypotonic cell swelling were studied in Ehrlich ascites tumour cells using the whole-cell recording mode of the patch-clamp technique. Currents were measured in the absence of added intracellular Ca2+ and with strong buffering of Ca2+. K+ current activated by cell swelling was measured as outward current at the Cl- equilibrium potential (ECl) under quasi-physiological gradients. It could be abolished by replacing extracellular Na+ with K+, thereby cancelling the driving force. Replacement with other cations suggested a selectivity sequence of K+ > Rb+ > NH4 approximately Na+ approximately Li+; Cs+ appeared to be inhibitory. The current-voltage relationship of the volume-sensitive K+ current was well fitted with the Goldman-Hodgkin-Katz current equation between -130 and +20 mV with a permeability coefficient of around 10(-6) cm s(-1) with both physiological and high-K+ extracellular solutions. The class III antiarrhythmic drug clofilium blocked the volume-sensitive K+ current in a voltage-independent manner with an IC50 of 32 microM. Clofilium was also found to be a strong inhibitor of the regulatory volume decrease response of Ehrlich cells. Cell swelling-activated K+ currents of Ehrlich cells are voltage and calcium insensitive and are resistant to a range of K+ channel inhibitors. These characteristics are similar to those of the so-called background K+ channels. Noise analysis of whole-cell current was consistent with a unitary conductance of 5.5 pS for the single channels underlying the K+ current evoked by cell swelling, measured at 0 mV under a quasi-physiological K+ gradient.

Ehrlich's ascites fluid adsorbed over protein A containing Staphylococcus aureus Cowan I produces inhibition of tumor growth.

Our earlier studies have shown that removal of various blocking factors from the sera of tumor-bearing animals and humans by adsorption over heat-attenuated and formalin-fixed-Staphylococcus aureus Cowan I (SAC) containing Protein A (PA) causes antitumor immune response. It was also shown that this procedure caused regression of a wide variety of established animal and human tumors. In the present investigation, the therapeutic potential of inoculation of ascites fluid adsorbed in vitro over non-viable SAC containing PA has been demonstrated in Ehrlich' s ascites tumor (EAT) in mouse. The antitumor effect was evident by a significant decrease in body weight (p<0.001) as well as significant reduction in viability of ascites tumor cells (p<0.001) in peritoneal cavity. However, some of the responding animals died earlier than controls, this may be due to the toxicity associated with therapy. The toxic effects were evident in decreased contents of glutathione, and increased activity of glutathione-S-transferase, decreased activity of microsomal enzymes and also in an early death of some of tumor regressed animals. The probable causes of toxicity of the therapy and prospects of reversing these toxic effects are discussed.

pH sensitive properties of Tc(V)-DMS: analytical and in vitro cellular studies.

Numerous clinical studies with the pentavalent technetium complex of dimercaptosuccinic acid [Tc(V)-DMS] seem to indicate its new role in nuclear oncology. Thus, we questioned what properties of the Tc(V)-DMS molecule associate with its tumoral tissue accumulation. Because studies have reported tumor tissue to be more acidic than normal tissue, acidification might be related to the Tc(V)-DMS localization in tumor tissue. Thus, in the present study, a working hypothesis drew to test the acidification as a plausible factor, and various analytical methods and an in vitro cellular system using Ehrlich ascites tumor cells (EATC) implemented. Analytical methodologies demonstrated the decrease of the overall negative charge of the Tc(V)-DMS molecule, promoted by the acidification of the analytical medium and the sample dilution. In the in vitro cellular experiment, acidification alone showed no effect on the radioactivity accumulation in EATC; nevertheless, if accompanied by a pre-dilution of the Tc(V)-DMS sample added into the cell incubation media, cellular radioactivity accumulation was observed. Thus, acidification as a mediator for the Tc(V)-DMS accumulation in tumoral cells, concurrently with dilution as the promoter of the process, constituted the foundation for discerning the working hypothesis.

A multifunctional protein: involvement of the alpha-1 serum protease inhibitor in SDS and high salt-stable DNA-protein complexes.

Occasionally new and intriguing roles arise for proteins with well established functions. The alpha-1 serum protease inhibitor (alpha-1 PI) represents another example. Sequence identities exist in the alpha-1 PI and in a nuclear 52-kDa glycoprotein which is involved in resistant DNA-polypeptide complexes. The results of Western blots support the identity of the two proteins and immunocytochemical studies indicate the nuclear location of the alpha-1 PI. Consistently, e.g. Ehrlich ascites tumor cells express the alpha-1 PI, and the fusion protein between the alpha-1 PI and the green fluorescent protein from Aequorea victoria shows intracellular accumulation and partly nuclear location.

Annexins from Ehrlich ascites cells inhibit the calcium-activated chloride current in Xenopus laevis oocytes.

The effect of annexins II, III and V, purified from different species, on the calcium-activated chloride current across the stage-V to stage-VI Xenopus laevis oocyte membrane was tested either directly, using calcium entry mediated by depolarization, by A23187 permeabilization of oocytes or indirectly by quisqualate stimulation of a metabotropic glutamate receptor in the membrane expressed by the oocyte after injection of mRNA. The annexins isolated from the Ehrlich ascites cell, which is a mouse tumor cell, were found to be potent inhibitors of the chloride current, showing half-maximal inhibition at 50 nM, whereas no block was found using bovine or porcine annexins isolated from lung tissue. Of the annexins tested, we found annexin III to be naturally occurring in the oocyte, while only trace amounts of annexins II and V could be demonstrated. The inhibition pattern varied somewhat according to the stimulus method, the inhibition being more complete when an indirect stimulus via the metabotropic receptor was applied compared to a direct calcium stimulus.

Analysis of phosphofructokinase subunits and isozymes in ascites tumor cells and its original tissue, murine mammary gland.

Phosphofructokinase (PFK) subunits and isozymes have been examined in ascites tumor cells and murine mammary gland, the tissue from where this tumor originated. Ascites tumor was found to contain predominantly the C-type subunit, whereas the L-type subunit was more abundant in mammary gland. An altered M-type subunit of lower electrophoretic mobility was found in both cell types and no M4 homotetramers were detected in either of them. Characteristic regulatory properties of ascites tumor PFK, i.e. insensitivity to fructose-1,6-P2 activation and inhibition by P-enolpyruvate, were also observed in the mammary gland isozyme. The nature of these properties and the contribution of the distinct subunit types to fructose-1,6-P2 activation are discussed.

Simple and rapid chromatographic method for the separation of major classes of ribosomal ribomononucleotides.

We have described a simple and rapid chromatographic method for the analytical and preparative separation of major types of ribosomal ribomononucleotides with Dowex 1-X10 (HCOO-, 37-74 microns) and Dowex 2-X10 (HCOO-, 37-74 microns) columns, by desorption with formiate solutions in 1-2 h. The separation has been achieved for Cp, Ap, Up and Gp, while a mixture of 2'-, and 3'-nucleoside phosphates desorbs as a single peak; with both resins, a successful separation was achieved with a load from 25 micrograms to 1 mg of ribomononucleotide mixture per ml of packed resin. A complete separation was achieved with Dowex 1, while the separation with Dowex 2 resin was even better. The resins cannot separate unusual nucleosides; therefore, our method is suitable for studies of ribonucleic acids with a low content of unusual nucleosides. Our method has been applied for the quantitative determination of the ribomononucleotide composition of 18S and 28S rRNAs, isolated from mammalian tissues: rat liver, mouse kidney and Ehrlich ascites cells. Dowex 1 and Dowex 2 resins afforded similar or identical ribomononucleotide compositions in all cases; analytical data were in agreement with the literature data. Our method is competitive, in several respects, with modern HPLC techniques for the separation of ribomononucleotides.

The effects of theanine, as a novel biochemical modulator, on the antitumor activity of adriamycin.

We studied the effects of theanine, a component of green tea leaves, on the antitumor activity of adriamycin (ADR) from the biochemical modulation view point. In vitro, theanine inhibited the ADR efflux from Ehrlich ascites carcinoma cells and maintained the ADR concentration in tumor cells. Theanine enhanced the inhibitory effect of ADR on tumor growth by 2.1-fold in vivo, and increased 2.9-fold the ADR concentration in the tumor, compared to the ADR alone group. An increase in ADR concentration was not observed in normal tissues, such as the heart and liver. Theanine did not enhance, rather tended to normalize the increase of lipid peroxide level and reduction of glutathione peroxidase activity as indicators of the ADR-induced side toxicity.

DNA cell cycle analysis of K562 and Ehrlich tumor cells by flow cytometry.

Monitoring cell activation processes and cell cycle progression in cells of various ontogenetic and phylogenetic origins is one of the base challenges in cellular biology. Flow cytometric analysis by DNA histograms show the cell cycle distribution and indicate the S-phase fractions of cell populations, as well as those cells in Go/G1 and G2/M phases. Human and murine normal diploid cells, K562 tumor cells (human origin) and Ehrlich cells (murine origin) were analysed using a propidium iodide staining procedure for flow cytometry. Our preliminary results suggested that this method may be a useful tool to detect cell populations with quantitative changes in DNA content, and to estimate cell fractions.

Characterization of gangliosides from Ehrlich ascites tumour cells and their variants.

Differences in the nature of the gangliosides present in two types of Ehrlich ascites tumour (EAT) cells, the adherent and non-adherent EAT cells, were studied. Gangliosides were isolated by DEAE Sephadex column chromatography and analysed by high-performance thin-layer chromatography (HPTLC). The non-adherent EAT (na-EAT) cells which grow in the peritoneal cavity of mice were selected for growth on basement membrane and tissue culture plastic to give the adherent EAT (a-EAT) cells. na-EAT cells contained 1.57 nmol lipid-bound sialic acid per mg protein and at least 12 different gangliosides, including major gangliosides such as GM3, GM2, GM1, GD3, GD1a and GT1b. On the other hand, the ganglioside pattern of a-EAT cells differed significantly from that of na-EAT cells, both quantitatively and qualitatively. The content of lipid-bound sialic acid in a-EAT cells was only 0.24 nmol per mg of protein. The gangliosides in a-EAT cells were characterized as GD1a and trisialogangliosides and, significantly, a-EAT cells did not contain monosialogangliosides. Neutral glycolipids were isolated from both cell lines and their patterns were compared. In contrast to the gangliosides pattern, their neutral glycolipid patterns were similar. Glucosylceramide and lactosylceramide were the major components in both types of cells. In addition to na- and a-EAT cells, a-EAT cells were passaged in mice by intraperitoneal injection, giving rise to a third variant (c/m EAT cells). We analysed the gangliosides in c/m EAT cells to determine whether there was a change in the ganglioside pattern found in na-EAT cells. After repeated passage of c/m EAT cells in mice, the pattern of gangliosides shifted to that of na-EAT cells. Alterations of ganglioside composition may be associated with the growth environment of the murine peritoneal cavity; alternatively, a selection process may have occurred.

Cloning and sequencing of the monocarboxylate transporter from mouse Ehrlich Lettré tumour cell confirms its identity as MCT1 and demonstrates that glycosylation is not required for MCT1 function.

Lactate transport is mediated in most tissues by H+-monocarboxylate-- cotransporters (MCTs). We have cloned and sequenced the lactate transporter from Ehrlich Lettré tumour cells by using the polymerase chain reaction (PCR) to amplify MCT1-related sequence from cDNA. The sequence is 93% and 87% identical to MCT1 from Chinese hamster and human respectively and so represents mouse MCT1. Most differences between MCT1 from Chinese hamster and mouse are conservative substitutions, located in hydrophilic parts of the molecule. Specific antipeptide antibodies confirm the presence of MCT1 protein in membranes from Ehrlich Lettré tumour cells. One difference between the mouse and Chinese hamster MCT1 is the absence of a predicted external consensus sequence for N-linked glycosylation in the mouse sequence. Using N-glycanase-F treatment and an in vitro translation system, we provide evidence that this glycosylation site is not actually utilised in Chinese hamster MCT1. These results are discussed in relation to current understanding of the roles of glycosylation of membrane proteins.

Identification of the integrin alpha 3 beta 1 as a component of a partially purified A-system amino acid transporter from Ehrlich cell plasma membranes.

We have previously reported [McCormick and Johnstone (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7877-7881] the partial purification of the Na(+)-dependent A-system amino acid transporter from Ehrlich cell plasma membranes and have suggested that a 120-130 kDa peptide, a major component of the purified fraction [octyl glucoside (OG) extract], is involved in Na(+)-dependent amino acid transport. In the present study, N-terminal sequence analysis of the 120-130 kDa peptide revealed a sequence similar to that of the alpha 3 subunit of the integrin alpha 3 beta 1. The presence of alpha 3 beta 1 was confirmed by Western blots of the OG extract probed with anti-alpha 3 or -beta 1 antibodies. Western blots also showed that an antibody originally raised against the 120-130 kDa peptide crossreacts with both the alpha 3 and beta 1 integrin subunits. Co-purification of alpha 3 beta 1 and Na(+)-dependent transport activity suggested that the two activities might be associated. Evidence that alpha 3 plays a role in transport is shown by the fact that an antibody against human alpha 3, but not beta 1, removed transport activity (approximately 25% loss) from cholate-solubilized Ehrlich membranes. Further purification of OG extracts using concanavalin A and wheat-germ lectin columns resulted in the separation of transport activity from the bulk (but not all) of alpha 3 beta 1 integrin without loss of the transport activity. These results indicate that the integrin itself is not essential for amino acid transport. Reconstitution of a purified alpha 3 beta 1-depleted protein fraction showed high levels of Na(+)-dependent, alpha-methylaminoisobutyric-acid-inhibitable amino acid transport in proteoliposomes, whereas reconstituted integrin alone showed little transport activity. However, in the integrin-depleted fractions, high amino acid uptake occurred in K+ which compromised the accurate measurement of the Na(+)-dependent component of uptake. The data suggest that alpha 3 may be associated with the A-system transporter and may modulate the activity of this carrier. Moreover, transfection of K562 and RD cells with human alpha 3 and alpha 2 cDNA showed that the former but not the latter increased A-system transport, thus providing more direct evidence that alpha 3 may modulate A-system transport activity.

Subcellular distribution of terminal alpha-D- and beta-D-galactosyl residues in Ehrlich tumour cells studied by lectin-gold techniques.

We have studied by high resolution in situ light and electron microscopic lectin-gold techniques the subcellular distribution of alpha-D-Gal residues using the Griffonia simplicifolia I-B4 isolectin and compared it with that of beta-D-Gal residues as detected with the Datura stramonium lectin in Ehrlich tumour cells grown as ascites or monolayer. The microvillar but not the smooth plasma membrane regions were labelled with the Griffonia simplicifolia I-B4 isolectin whereas both plasma membrane regions were equally well labelled with the Datura stramonium lectin. Elements of the endocytotic/lysosomal system such as coated membrane invaginations and vesicles, early and late endosomes and secondary lysosomes were positive for both alpha-D-Gal and beta-D-Gal residues. A particular feature of Ehrlich tumour cells is an elaborate tubular membrane system located in the pericentriolar region which is labelled throughout by both lectins and represents part of the endosomal system. In the Golgi apparatus labelling with both lectins was observed to commence in trans cisternae which is indirect evidence for a joint distribution of the sequentially acting beta 1,4 and alpha 1,3-galactosyl-transferases.

Spontaneous overexpression of heat-shock proteins in Ehrlich ascites carcinoma cells during in vivo growth.

Ehrlich carcinoma (EC) cells isolated from mice at different phases of ascites growth were exposed to hyperthermia (44 degrees C), or oxidative stress (hydrogen peroxide or vikasol), or ATP depletion induced by rotenone. These exposures caused protein aggregation and rapid necrotic death in exponentially growing EC cells. On the contrary, the same cell culture at stationary phase of growth became considerably more resistant to all the above cytotoxic treatments, and the level of aggregated protein was significantly lower in stressed stationary EC cells than that in exponential ones. Comparative immunoblotting has revealed the unexpected expression of inducible 70 kDa heat-shock protein form (HSP68), as well as accumulation of HSP27 and HSP90 in the thermo- and drug-resistant stationary EC cells. It is suggested that the in vivo occurring HSP overexpression in stationary EC cells is an adaptive modulation of the tumor cell phenotype to maintain the viability of ascites EC cells under chronic deficiency of oxygen and nutrients.