Enhanced antitumour activity of doxorubicin and simvastatin combination loaded nanoemulsion treatment against a Swiss albino mouse model of Ehrlich ascites carcinoma.

Doxorubicin (DOX) is the most commonly used anticancer drug; however, it has limited use because prolonged administration may result in severe cardiotoxicity. Simvastatin (SIM), generally prescribed for hypercholesterolaemia, has also shown salubrious results in the monotherapy or combinational drug therapy of different cancers in various models. Nanoparticle drug delivery systems are a novel way of improving therapeutics and also improving the absorption and specificity of drugs towards tumour cells. In this study, we exploited this technology to increase drug specificity and minimize imminent adverse effects. In this study, the antitumour activity of the combination formulas of DOX and SIM, either loaded in water (DOX-SIM-Solution) or nanoemulsions (NEs) (DOX-SIM-NE), was evaluated in a Swiss albino mouse model of Ehrlich ascites carcinoma. The anticancer effect was assessed by quantifying the change in body weight, mean survival time, and percent increase in lifespan (%ILS), determining haematological and serum biochemical parameters (liver function test, kidney function test and lipid profile parameters) as well as studying the histopathological alterations in liver tissues. We observed a clear increase in %ILS of the DOX-SIM-Solution group (265.30) that was double the %ILS of the DOX-SIM-NE group (134.70). However, DOX-SIM-NE had a non-toxic effect on the haematological parameters, whereas DOX-SIM-Solution increased the levels of haemoglobin and lymphocytes. Furthermore, the encapsulation of SIM and DOX into NEs improved the levels of all serum biochemical parameters compared to the DOX-SIM-Solution. A reduction in the side effects of DOX-SIM-NE on the liver was also established using light microscopy, which revealed that the morphologies of the hepatocytes of the mice were less affected by administration of the DOX-SIM-NE treatment than with the DOX-SIM-Solution treatment. The study showed that incorporating SIM into the DOX-loaded-NE formulation remarkably improved its efficiency and simultaneously reduced its adverse effects.

Toxicity and antitumor potential of Mesosphaerum sidifolium (Lamiaceae) oil and fenchone, its major component.

BACKGROUND: The essential oil from Mesosphaerum sidifolium (L'Hérit.) Harley & J.F.B.Pastore (syn. Hyptis umbrosa), Lamiaceae (EOM), and its major component, have been tested for toxicity and antitumor activity. METHODS: EOM was obtained from aerial parts of M. sidifolium subjected to hydro distillation, and gas chromatography-mass spectrometry was used to characterize the EOM chemical composition. The toxicity was evaluated using haemolysis assay, and acute toxicity and micronucleus tests. Ehrlich ascites carcinoma model was used to evaluate the in vivo antitumor activity and toxicity of EOM (50, 100 and 150 mg/kg), and fenchone (30 and 60 mg/kg) after 9 d of treatment. RESULTS: The EOM major components were fenchone (24.8%), cubebol (6.9%), limonene (5.4%), spathulenol (4.5%), ß-caryophyllene (4.6%) and α-cadinol (4.7%). The HC50 (concentration producing 50% haemolysis) was 494.9 µg/mL for EOM and higher than 3000 µg/mL for fenchone. The LD50 for EOM was approximately 500 mg/kg in mice. The essential oil induced increase of micronucleated erythrocytes only at 300 mg/kg, suggesting moderate genotoxicity. EOM (100 or 150 mg/kg) and fenchone (60 mg/kg) reduced all analyzed parameters (tumor volume and mass, and total viable cancer cells). Survival also increased for the treated animals with EOM and fenchone. For EOM 150 mg/kg and 5-FU treatment, most cells were arrested in the G0/G1 phase, whereas for fenchone, cells arrested in the S phase, which represents a blockage in cell cycle progression. Regarding the toxicological evaluation, EOM induced weight loss, but did not induce hematological, biochemical or histological (liver and kidneys) toxicity. Fenchone induced decrease of AST and ALT, suggesting liver damage. CONCLUSIONS: The data showed EOM caused in vivo cell growth inhibition on Ehrlich ascites carcinoma model by inducing cell cycle arrest, without major changes in the toxicity parameters evaluated. In addition, this activity was associated with the presence of fenchone, its major component.

Anti-cancer and cardioprotective effects of indol-3-carbinol in doxorubicin-treated mice.

Doxorubicin (DOX) is a broad-spectrum antitumor antibiotic used in treatment of cancer. Its effect may be complicated by increased risk of cardiotoxicity. It was suggested that natural compounds with anticancer properties can be used in combination with DOX to decrease its dose and side effects. Indole-3-carbinol (I3C) is one of the phytochemicals that was shown to have anti-cancer effect. Our aim was to detect the possible chemosensitizing effects of I3C in DOX-induced cytotoxicity and the possible cardioprotective effects of I3C in DOX-induced cardiotoxicity. One hundred mice were divided into five equal groups: Control untreated group, solid Ehrlich carcinoma (SEC), SEC + DOX, SEC + I3C, SEC + DOX + I3C. Tumor volume, serum creatinine kinase and lactate dehydrogenase were measured. Also, tissue malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), sphingosine kinase-1 (SphK1) activity and interleukin-6 (IL-6) were determined. Parts of the tumor and cardiac tissues were subjected to histopathological examination. DOX or I3C alone or in combination induced significant increase in tumor CAT and SOD with significant decrease in tumor volume, tumor MDA, SphK1 activity and IL-6 and alleviated the histopathological changes with significant increase in the apoptotic index and significant decrease in tissue bcl2 compared to SEC group. Also, DOX induced cardiotoxicity which was ameliorated by I3C. In conclusion, DOX/I3C combination had a better effect than each of DOX or I3C alone against SEC in mice with marked improvement of the cardiotoxicity induced by DOX.

Effect of electromagnetic microwave radiation on the growth of Ehrlich ascites carcinoma.

Daily exposure of mouse recipients of Ehrlich ascites carcinoma to electromagnetic radiation of the microwave range leads to a change in the dynamics of tumor growth by decreasing the total number of cells. The number of tumor cells with blebbing morphological signs after microwave radiation increases gradually with tumor growth. The maximum content of tumor cells in the state of blebbing is observed during active proliferation in tumor-recipient mice of the control group (without irradiation).

Odor cues released by Ehrlich tumor-bearing mice are aversive and induce psychological stress.

BACKGROUND/AIMS: This study aimed to verify if odor cues released by Ehrlich tumor-bearing mice are aversive and stressful. METHODS: Female mice were divided into a control group and an experimental group. One animal of each experimental pair of mice was inoculated with 5 × 10(6) Ehrlich tumor cells intraperitoneally; the other animal was kept undisturbed and was referred to as a CSP (companion of sick partner). One mouse of each control pair was treated intraperitoneally with 0.9% NaCl (1 mg/kg); the other animal (CHP, companion of healthy partner) was kept undisturbed. RESULTS: It was shown that, in relation to CHP, CSP mice (1) spent less time within the companion zone in a T-maze place preference test, (2) had increased levels of social interaction, (3) had increased levels of plasmatic adrenaline and noradrenaline and (4) displayed no changes in serum corticosterone levels before and after an immobilization stress challenge. It was also shown that (5) cohabitation with 2 tumor-bearing mice was more effective in decreasing neutrophil oxidative burst than cohabitation with 1 sick partner and (6) the presence of a healthy conspecific within the cage of the tumor-injected/CSP pair abrogated the effects of cohabitation on neutrophil activity. These results show that odor cues released by Ehrlich tumor-injected mice are aversive and induce psychological stress. CONCLUSION: We postulate that the aversive response induced by the chemosignals released by Ehrlich tumor-injected animals activates the sympathetic nervous system and causes the neuroimmunal changes that occur in the mice cohabiting with the sick mice.

Thymus in experimental carcinogenesis of the prostate gland.

We studied structural changes in the prostate gland, thymus, and lymph nodes in CBA mice after transplantation of Ehrlich ascites tumor cells into the prostate gland. On experimental day 5, the number of blood and lymph vessels decreased in the gland; the percentage of connective tissue elements and glandular tissue and the number of immunoblasts in the thymus increased. On day 18, the number of blood vessels in the tumor decreased; the width of the cortex and glandular tissue increased in the thymus, while the number of immunoblasts was reduced. On day 28, tumor infiltration and increased number of lymphatic vessels in its stroma were observed; parenchyma was reduced, and the area of the connective tissue increased in the thymus. These structural changes indicated the development of accidental involution of the thymus during carcinogenesis of the prostate.

Apoptogenic effects of Tricholoma giganteum on Ehrlich's ascites carcinoma cell.

This study explored the efficacy of Fa fraction of Tricholoma giganteum against Ehrlich's ascites carcinoma (EAC). Mechanisms of apoptogenic effect of the fraction were delineated. The flow cytometric analysis of EAC cells, showed an increase in number of cells in sub-G(0)/G(1) population and reduction in the G(2)/M phase due to the treatment thus suggesting apoptosis. The induction of apoptosis has also been confirmed by nuclear staining that demonstrated distinctive morphological features of apoptosis. Our data also revealed an increase in the expression of pro-apoptotic protein p53 in EAC and induced factors contributing to apoptosis. Pro-apoptotic gene Bax was up-regulated during p53-mediated apoptosis. No significant change in the expression of anti-apoptotic protein Bcl-2 was observed ensuing in decrease of the Bcl-2/Bax ratio. p53-mediated growth arrest involves p21 as a major effecter, which interestingly showed moderate elevation. All these observations indicate that Fa fraction of T. giganteum induces apoptogenic signal in EAC.

Combinatorial antitumor effect of naringenin and curcumin elicit angioinhibitory activities in vivo.

Curcumin has long been used as an antioxidative, antiinflammatory, and modulator of pathological angiogenesis, whereas naringenin is a well-known immunomodulator. In this report, we investigated the effect of curcumin and naringenin on the growth of Ehrlich ascites carcinoma tumor model. To achieve this, Swiss albino mice were implanted intraperitoneally with 1 × 106 Ehrlich ascites carcinoma cells followed by the administration of oral doses of naringenin and curcumin either individually (50 mg/kg body weight) or in combination (20 mg/kg body weight each). A marked reduction has been seen in the total number of cells (80%) and accumulation of ascetic fluid (55%) when these drugs were administered together. These drugs proved to be an effective angio-inhibitory compound and confirmed by different in vivo assay systems, viz. peritoneal/skin angiogenesis and chorioallantoic membrane assay. Antiangiogenic and antiproliferative effect of these compounds alone or in combination was further corroborated with immunoblot results where we confirmed the downregulation of vascular endothelial growth factor, Hif1α, heat shock protein 90, and p-Akt. Furthermore, treatment with naringenin and curcumin alone or in combination substantially improved hepatocellular architecture and no noticeable neoplastic lesions or cellular alteration were reported. These outcomes put forward a plausible clinical application of these diet-derived compounds, as both angioinhibitory and antitumor in association with conventional therapy.

Effect of perfluorodecalin on viability of Ehrlich ascites tumor cells under conditions of hypoxia.

Perfluorodecalin increased survival rate of Ehrlich ascites tumor cells under pathological conditions of hypoxia in combination with hyperkalemia. High potassium medium increased the content of lysophospholipids in samples, while in the presence of perfluorodecalin, phosphatidylethanolamine level decreased.

Macrophage-melanoma cell heterokaryons. IV. Unmasking the macrophage-specific membrane receptor.

Mouse peritoneal macrophages possess a specific plasma membrane receptor for antibody-coated particles. Sheep red cells coated with rabbit 7S antibody attach readily to the macrophage surface and are subsequently interiorized. The fusion of macrophage with nonphagocytic mouse melanoma cells produces heterokaryons in which the macrophage receptor is drastically altered. The receptor is present shortly after fusion and heterokaryons are actively phagocytic. The ability to bind and ingest red cells is, however, progressively lost over the next 12-24 hr and does not reappear thereafter. Exposure of heterokaryons to trypsin (1-100 microg/ml for 30 min at 37 degrees C) results in the reappearance of initial receptor activity and the unmasking of the surface receptor. This property is again lost upon subsequent cultivation. The masking process takes place when cells are cultivated in the absence of IgG so that the adsorption of antibody from the medium is not responsible for this phenomenon. Inhibition of heterokaryon protein synthesis preserves phagocytic activity in a reversible fashion and prevents the masking of macrophage receptors. Inhibition of melanoma RNA synthesis before fusion is also able to block subsequent masking, but is ineffective if delayed until after fusion. Ultraviolet irradiation of the melanoma cell before fusion prevents subsequent masking, whereas similar treatment of the macrophage has no effect. Cells differ markedly in their ability to mask the macrophage phagocytic receptor after fusion. Ehrlich ascites tumor cells mask the receptor rapidly, primary chick fibroblasts minimally, and embryonic chick erythrocytes not at all.

31P nuclear magnetic resonance evidence for the regulation of intracellular pH by Ehrlich ascites tumor cells.

The phenomenon of intracellular pH (pHin) regulation in cultured Ehrlich ascites cells was investigated using 31P nuclear magnetic resonance (NMR) spectroscopy. Measurements were made with a Bruker WH 360 wide bore NMR spectrometer at a 31P frequency of 145.78 MHz. Samples at a density of 10(8) cells ml-1 were suspended in a final volume of 2 ml of growth medium in 10 mm diameter NMR tubes. Intracellular pH was calculated from the chemical shifts of either intracellular inorganic phosphate (Piin) or intracellular 2-deoxyglucose-6-phosphate (2dG6Pin). The sugar phosphate was used as a pH probe to supplement the Piin measurements, which could not always be observed. When available, the pHin calculated from the Piin peak was identical within experimental error to the pHin calculated from the 2dG6Pin peak. Intracellular pH was measured to be more alkaline than the medium at an external pH (pHex) below 7.1. Typical values were pHin = 7.00 for pHex = 6.50. These measurements were constant for times up to 165 min using well-energized, respiring cells. This pH gradient was seen to collapse immediately upon onset of anaerobic shock. Above a pHex of 7.2 there was no significant difference between pHin and pHex. These results unequivocally demonstrate the steady state nature of the pH regulation and its dependence upon energization.

Antiangiogenic and proapoptotic activities of allyl isothiocyanate inhibit ascites tumor growth in vivo.

The authors investigate the antiangiogenic and proapoptotic effects of mustard essential oil containing allyl isothiocyanate (AITC) and explore its mechanism of action on Ehrlich ascites tumor (EAT) cells. Swiss albino mice transplanted with EAT cells were used to study the effect of AITC. AITC was effective at a concentration of 10 mum as demonstrated by the inhibition of proliferation of EAT cells when compared with the normal HEK293 cells. It significantly reduced ascites secretion and tumor cell proliferation by about 80% and inhibited vascular endothelial growth factor expression in tumor-bearing mice in vivo. It also reduced vessel sprouting and exhibited potent antiangiogenic activity in the chorioallantoic membrane and cornea of the rat. AITC arrested the growth of EAT cells by inducing apoptosis and effectively arrested cell cycle progression at the G1 phase. The results clearly suggest that AITC inhibits tumor growth by both antiangiogenic and proapoptotic mechanisms.

Apoptosis-inducing effects of Morinda citrifolia L. and doxorubicin on the Ehrlich ascites tumor in Balb-c mice.

Morinda citrifolia L. (Noni) is a herbal remedy with promising anti-cancer properties. However, its effects on various cancers are to be investigated to make a firm conclusion before implementing it into the clinical practice. Therefore, we investigated the cytotoxic potential of noni on Ehrlich ascites tumor grown in female Balb-c mice and also combined it with a potent anti-cancer agent, doxorubicin. One group received noni only (n = 8), another one doxorubicin (n = 8), and the other one noni + doxorubicin (n = 8) for 14 days after the inoculation of cells. The control group (n = 7) received 0.9% NaCl only. We found that short and long diameters of the tumor tissues were about 40-50% smaller, compared to those in control group. This anti-growth effect resulted from the induction of apoptosis, which was confirmed by the positive results from the Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) analysis and the active caspase-3 cells in tissues. Apoptosis also confirmed by caspase-cleaved cytokeratin 18 elevation in serum of the treated groups. Further, the proliferation was decreased, which was immunohistochemically shown by the PCNA staining. We conclude that noni may be useful in the treatment of breast cancer either on its own or in combination with doxorubicin. Further studies are warranted to assess the dosage and safety of using noni fruit juice in conjuction with anti-cancer drugs against breast cancer.

[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.

Tumor associated release of interleukin-10 alters the prolactin receptor and down-regulates prolactin responsiveness of immature cortical thymocytes.

The soluble factors produced either by Ehrlich's ascites carcinoma (EAC) or thymic adherent cells (TAC) of tumor-bearing mice comprising of CD11b(+) and CD11c(+) antigen-presenting cells caused a sharp decrease in prolactin (PRL)-induced ConA-mediated effect on survival of PNA(+) thymocytes. Similar suppression of PRL-induced effect was observed when the cells were cocultured with TAC of EAC-bearing mice. Anti-IL-10 antibody could reverse the PRL inability to induce ConA-mediated effect on PNA(+) thymocyte survival, indicating the presence of IL-10 in EAC culture supernatant (EAC sup) and thymic microenvironment. IL-10 could block PRL-induced proliferation of PNA(+) thymocytes without affecting spontaneous apoptosis. IL-10 altered the expression of the long-form (LF) of PRL-R and reduced the PRL binding of the cells, suggesting down-regulation of the PRL effect on PNA(+) thymocyte by the cytokine. Induction of tumor, which was found to increase the IL-10 secretion by TAC, also modified the PRL-R (LF) to PRL-R (SF). Since PRL plays a role in survival, proliferation and differentiation of lymphoid progenitor cells, the tuning of PRL action by IL-10 may be a possible mechanism of depletion of immature cortical thymocytes and thymic atrophy in tumor-bearing mice.

Nutrition of mouse ascites tumor cells in primary culture. II. Specific requirement for glucose.

Primary monolayer cultures were prepared from mouse ascites tumor cells of the Ehrlich-Lettré line and cultivated in chemically defined media containing various carbohydrates as the sole energy source. An absolute glucose requirement for culture survival was found, which could be satisfied by either the alpha or beta forms. The cultures died within 2-3 days when D-mannose, D-fructose, or D-galactose was substituted for D-glucose. Of over 50 other carbohydrates and related compounds tested, none could replace glucose. Cells of the Ehrlich and Ehrlich-Lettré lines suspended in balanced salt solutions containing various carbohydrates removed D-glucose, D-mannose, D-fructose, D-glucosamine, and 2-deoxy-D-glucose from the suspending fluid. In the presence of D-glucose, culture survival was inhibited by D-mannose, D-fructose, L-glucose, and D-galactose. D-Glucosamine and 2-deoxy-D-glucose strongly inhibited culture survival with the 50% inhibition points at 375 mg/liter and 62.5 mg/liter, respectively. The inhibition by D-glucosamine could be reversed by alpha- and beta-glucose, but not by D-mannose or D-fructose. The inhibition by 2-deoxy-D-glucose could be reversed completely by alpha- and beta-glucose, to a slight degree by D-mannose, and not at all by D-fructose.

Pathogenesis of ascites in mice with peritoneal carcinomatosis.

For clarification of the mechanisms underlying formation of malignant ascites, alterations in lymphatic transport from the peritoneal cavity and in peritoneal capillary permeability to protein were studied sequentially in mice inoculated ip with Ehrlich-Lettre ascites tumor cells. All animals developed detectable ascites within 5-7 days of the injection. Diaphragmatic and retrosternal lymph vessels became radiopaque within 30 hours of the ip injection of radiopaque contrast material in control animals without ascites and in 12 experimental mice receiving contrast material 1-3 days after injection of tumor cells. No lymph vessels were opacified in 3 of 4 animals when contrast material was injected on day 5 or in any animal receiving contrast material on or after day 7 following the tumor cell injection. We determined alterations in peritoneal capillary permeability in the second group by measuring the concentration of iv injected Evans blue dye in eluates of sections of peritoneum and contiguous underlying tissue removed 3 hours after injection of dye. Permeability averaged 1.5 times normal (P = 0.02) by day 3 after tumor cell injection, 2 times normal by day 5, and 3 times normal by day 7; it remained at the final level. Although lymph drainage became impaired within 24 hours of the detection of ascites, a progressive increase in capillary permeability began 2 days earlier and was probably the predominant alteration in pathogenesis of the effusion.

Antibody-dependent lysis of tumor cells in vivo. I. Early lysis of tumor cells.

Possible mechanisms of in vivo killing of tumor cells by antibody (Ab) were explored in a model in which Ab suppresses tumor growth in mice. A proportion of tumor cells injected into the peritoneal cavities of complement-deficient A/J mice was lysed within 30 minutes in the presence of anti-tumor cell Ab as demonstrated by the release of 51Cr in vivo. Experiments in which [125I]5-iodo-2'-deoxyuridine-labeled tumor cells were used showed that most tumor cells did not leave the injection site and that lysis occurred inside the peritoneal cavity. In addition, a proportion of the remaining sensitized tumor cells was significantly damaged, as shown by their decreased resistance to osmotic lysis. Morphologic studies ruled out phagocytosis of tumor cells as a mechanism of major significance. Tumor cell damage but not tumor cell lysis could also be induced in vitro with Ab and normal peritoneal cells. These results provided direct evidence of primary cytolysis induced by Ab in vivo, though other concomitant cytotoxic mechanisms may also take place in this model.

Increase in nonspecific adsorptive endocytosis in anthracycline- and vinca alkaloid-resistant Ehrlich ascites tumor cell lines.

Numerous biochemical and drug transport studies have focused on the role of the plasma membrane or membrane-associated processes in anthracycline and vinca alkaloid resistance. Cationized ferritin, which binds electrostatically to anionic sites on the plasma membrane, was used as a membrane marker in a morphometrical ultrastructural study. The nonspecific adsorptive endocytosis was significantly increased in Ehrlich ascites tumor cell lines resistant to daunorubicin (DNR), doxorubicin, vincristine, and vinblastine compared to that in sensitive cells. Thus cells resistant to DNR internalized 2.04% of their plasma membrane per minute, in contrast to 1.17% in sensitive cells. Because the measured cell surface areas in sensitive and resistant cells were unchanged during the experiments, increased membrane traffic (recycling) from intracytoplasmic compartments to the plasma membrane must have occurred in resistant cells. Possible implications of these findings include a resistance-linked phenotype without significance for resistance per se, an increase in membrane repair, and a connection between increased membrane traffic and increased active drug extrusion in resistant cells.

Mechanism of the linkage between te electrophoretic mobility and oxygen uptake of ascites tumor cells.

Electrophoretic mobility, glucose metabolism, and oxygen uptake were studied in three leukemic and in four nonleukemic strains of ascites tumor cells. The cells differed markedly in mobility. This variation was related neither to cell growth nor to rates of endogenous respiration and aerobic glycolysis. The nonleukemic tumor cells showed higher mobility than did the leukemic cells. Additional increases in mobility appear to be related to suppressed oxygen uptake, which results from the addition of glucose or 0.05 mM 2,4-dinitrophenol. The augmented negative surface charge does not seem to be related to those ionogenic sites susceptible to neuraminidase. However, RNase treatment of the nonleukemic tumor cell does reveal the presence in their surface membrane of negatively charged sites susceptible to this enzyme. Such RNase-susceptible ionogenic sites presumably redistribute at the cell surface membrane from the inner sites as a response to suppressed oxygen uptake. This results in a higher negative surface charge and increased mobility. The leukemic cells, on the other hand, did not show any change in oxygen uptake or mobility in the presence of glucose. Moreover, the reduced rate of oxygen uptake induced by the addition of 0.05 mM 2,4-dinitrophenol did not result in any significant alterations in mobility. This is consistent with the observation that the ionogenic sites susceptible to RNase do not appear at the surface of the leukemic cells upon suppressed oxygen uptake. The leukemic cells have thus been shown to differ from the nonleukemic tumor cells in certain aspects of their glucose metabolism as well as in the electrophoretic properties of their surface.