Ascites Tumor Growth Research Articles

Synthesis and Evaluation of 1-Benzhydryl-sulfonyl-piperazine Derivatives as Inhibitors of Tumor Growth and Tumor Angiogenesis of Mouse Ehrlich Ascites Tumor In Vivo

A series of novel 1-benzhydryl-sulfonyl-piperazine derivatives 3(a-e) were synthesized by nucleophilic substitution reaction of 1-benzhydryl-piperazine with different sulfonyl chlorides and were characterized by 1H NMR, LC/MS, FTIR and elemental analysis. In the present study, the compounds 3(a-e) exhibited in vivo inhibition of Ehrlich ascites tumor (EAT) cell growth and increased the Median Survival Time (MST) and %ILS of EAT bearing mice. Further treatment of derivatives in vivo resulted in reduction of EAT cell number and ascites formation. The efficacy of the derivatives to inhibit the angiogenesis in vivo was evaluated in tumor bearing mice peritoneum and chorio allantoic membrane (CAM) model. The compounds suppressed the blood vessel formation in vivo in mice peritoneum and in CAM. Among the compounds studied, 3e demonstrated highest tumor inhibitory and anti-angiogenic effects against mouse tumor. However, this phenomenon needs detailed investigation.

Inhibition of murine tumor development by the lectin wheat germ agglutinin.

The antitumor activity of lectins were examined by studying the effect of lectins on the in vivo growth of murine tumors. The lectin wheat germ agglutinin (WGA) inhibited the growth of MM46 ascites tumors in vivo as effectively as syngeneic antitumor antiserum, but other lectins were not effective in vivo. WGA also had an inhibitory effect on other syngeneic and allogeneic ascites tumors, a fact which is consistent with its wide range of target tumor cells demonstrated in the in vitro cytolysis system. These results suggest that WGA is a better antitumor agent than other plant lectins. The therapeutic effect of WGA might be, in part, due to its ligand activity between tumor cells and macrophages that induces lectin-dependent macrophage-mediated cytolysis, in addition to its direct cytostatic activity.

Antitumoural Effect of an l‐Amino Acid Oxidase Isolated from Bothrops jararaca Snake Venom

An L-amino acid oxidase (BjarLAAO-I) from Bothrops jararaca snake venom was highly purified using a stepwise sequential chromatography on Sephadex G-75, Benzamidine Sepharose and Phenyl Sepharose. Purified BjarLAAO-I showed a molecular weight around 60,000 under reducing conditions and about 125,000 in the native form, when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. BjarLAAO-I is a homodimeric acidic glycoprotein, pI approximately 5.0, and N-terminal sequence showing close structural homology with other snake venom LAAOs. The purified enzyme catalysed the oxidative deamination of L-amino acids, the most specific substrate being L-Phe. Five amino acids, L-Ser, L-Pro, L-Gly, L-Thr and L-Cys were not oxidized, clearly indicating a significant specificity. BjarLAAO-I significantly inhibited Ehrlich ascites tumour growth and induced an influx of polymorphonuclear cells, as well as spontaneous liberation of H(2)O(2) from peritoneal macrophages. Later, BjarLAAO-I induced mononuclear influx and peritoneal macrophage spreading. Animals treated with BjarLAAO-I showed higher survival time.

Glucose-dependent active ATP depletion by koningic acid kills high-glycolytic cells

Many types of cancer cells depend heavily on glycolysis for energy production even in aerobic conditions. We found that koningic acid (KA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), selectively kills high-glycolytic cells through glucose-dependent active ATP deprivation. Out of seven tumor cell lines tested, growth of six cell lines, which had high glycolytic capacity, was inhibited by KA, whereas three normal cell lines, which had low glycolytic activity, were insensitive to KA. The growth inhibition and caspase-independent cell death in sensitive cells were related to severe ATP depletion that was promoted by glucose phosphorylation. Although GAPDH was completely inhibited in KA-insensitive CHO-K1 cells, KA-mediated ATP depletion was less extensive and transient, possibly due to utilization of ketogenic essential amino acids as energy source. KA suppressed Ehrlich ascites tumor growth in vivo and benefited the survival of the affected mice.

Enhanced antitumor activity of irinotecan combined with propolis and its polyphenolic compounds on Ehrlich ascites tumor in mice

The effects of the anticancer drug irinotecan combined with ethanolic extract of propolis (EEP), a water-soluble derivate of propolis (WSDP), quercetin and naringin on the growth of Ehrlich ascites tumor (EAT) and the life span of tumor-bearing Swiss albino mice were studied. Test components were given to mice intraperitoneally ( i.p.) at doses of 100 mg kg −1 for three consecutive days before the i.p. injection of EAT cells (1 × 10 6). Irinotecan was administered i.p. at dose of 50 mg kg −1 on days 1, 13, and 19 after tumor cell inoculation. The results clearly demonstrate the synergistic action of irinotecan and EEP on survival time. These results suggest that clinical trials using a propolis preparation EEP combined with irinotecan may be beneficial in maximizing antitumor activity and minimizing post-chemotherapeutic reactions to the cytostatic drug.

Proapoptotic and antitumor activities of the HMG-CoA reductase inhibitor, lovastatin, against Dalton's Lymphoma Ascites tumor in mice

Background Diet rich in fat have a clear effect on the tumor incidence in humans. Increased level of lipid peroxidation were found in colon, liver, breast and kidney carcinogenesis. Although the beneficial effects of statins for cardiovascular diseases are well established, their importance in the area of cancer therapeutics has recently gained recognition. Many studies of lovastatin in in vitro systems and experimental animals have been reported as an effective antitumor agent. However, phase I/II clinical trials in cancer patients demonstrated a minor to non-significant responses. Hence more studies in different tumor models using doses corresponding to that used to reduce lipid in human are required to support the antitumor activity. Methods The antitumor activity was evaluated using Daltons' Lymphoma Ascites (DLA) cell line-induced ascites tumor model in mice. Proapoptotic activity was evaluated in DLA cell line induced ascites animals after the treatment of lovastatin. Apoptosis was analyzed morphologically by staining with Giemsa and biochemically by observing the laddering of DNA in agarose gel electrophoresis. In vitro cytotoxic activity of lovastatin was studied by trypan blue dye exclusion method. Lipid peroxidation inhibiting activity was demonstrated in Fe 2+-ascorbate induced rat whole liver homogenate. Results Lovastatin dose dependently inhibited the ascites tumor growth at 4 and 16 mg/kg body wt (p.o). The percentage increase in life span (%ILS) in the 16 mg/kg treated group was 61.8% ( P < 0.01). Single dose of lovastatin (16 mg/kg body wt, p.o) was also effective to accelerate the apoptosis in the ascites tumor bearing mice that was evident from the multiple fragmentation of DNA in gel electrophoresis. Further the morphological analysis of DLA cells aspirated from the lovastatin treated animals showed a significant ( P < 0.01) increase of apoptotic cells (15.5 ± 3%) than the control animals (6.5 ± 1%). Concentration of lovastatin required for the 50% of the cytotoxicity was 37 ± 5 μg/ml. Lovastatin at its low concentrations were effective to inhibit lipid peroxidation. Conclusions The antitumor activity of lovastatin against the ascites tumor is due to its proapoptotic and cytotoxic activities. Lovastatin at low concentrations inhibited Fe 2+ induced lipid peroxidation in in vitro system. The proapoptotic and lipid peroxidation inhibiting activities of the lipid lowering drug lovastatin may further suggest its possible therapeutic use as a cancer chemopreventive agent.

Intraperitoneal noxytiolin as an experimental chemotherapeutic agent.

Abstract The effect of intraperitoneal noxytiolin on the growth of Ehrlich's ascites tumour in mice has been studied. A dose of 1 g. per kg. body-weight produced a significant slowing of the growth of ascites tumours. However, systemic administration (subcutaneous) had no significant chemotherapeutic effect in this tumour model.

Polyphenolic compounds from propolis modulate immune responses and increase host resistance to tumour cells

Propolis contains a variety of polyphenolic compounds. We investigated the effect of a water-soluble derivatives of propolis (WSDP) and polyphenolic compounds, components of propolis, on growth of Ehrlich ascites tumour (EAT) in mice. Tumour in peritoneal cavity was produced by 2×106 EAT cells. WSDP and polyphenolic compounds (caffeic acid-CA, caffeic acid phenethyl ester-CAPE and quercetin-QU) were given to mice perorally (po). It was found that the volume of ascitic fluid induced by EAT cells and total number of cells present in the peritoneal cavity was markedly reduced in EAT-bearing mice treated with test components and the survival time of treated mice was prolonged. Inhibition of EAT growth was due to their effect on the immune system of mice. When innate and acquired immune responses were evaluated, a dose-related increase of cytotoxic T-cell, NK and B cells activity was observed in test components-treated mice. Furthermore, exposure of animals to test components increased functional activity of macrophages to produce factors regulating the function of B-, T-, and NK- cells respectively. In conclusion, these findings imply that the antitumour activity of WSDP and polyphenolic compounds of propolis enhanced host resistance in the EAT tumour model, increasing the activities of macrophages, cytotoxic T cells, B cells and NK cells.

Butyrate-induced proapoptotic and antiangiogenic pathways in EAT cells require activation of CAD and downregulation of VEGF

Butyrate, a short-chain fatty acid produced in the colon, induces cell cycle arrest, differentiation, and apoptosis in transformed cell lines. In this report, we study the effects of butyrate (BuA) on the growth of Ehrlich ascites tumor (EAT) cells in vivo. BuA, when injected intraperitoneally (i.p) into mice, inhibited proliferation of EAT cells. Further, induction of apoptosis in EAT cells was monitored by nuclear condensation, annexin-V staining, DNA fragmentation, and translocation of caspase-activated DNase into nucleus upon BuA-treatment. Ac-DEVD-CHO, a caspase-3 inhibitor, completely inhibited BuA-induced apoptosis, indicating that activation of caspase-3 mediates the apoptotic pathway in EAT cells. The proapoptotic effect of BuA also reflects on the antiangiogenic pathway in EAT cells. The antiangiogenic effect of BuA in vivo was demonstrated by the downregulation of the secretion of VEGF in EAT cells. CD31 immunohistochemical staining of peritoneum sections clearly indicated a potential angioinhibitory effect of BuA in EAT cells. These results suggest that BuA, besides regulating other fundamental cellular processes, is able to modulate the expression/secretion of the key angiogenic growth factor VEGF in EAT cells.

Mechanism of inhibition of ascites tumor growth in mice by curcumin is mediated by NF-kB and caspase activated DNase

One of the most clinically relevant biological activities of curcumin is its anti-cancer property, implicating multiple intracellular pathways in the process. In the present report, we investigated the effect of curcumin on the activation of apoptotic and anti-angiogenic pathways in Ehrlich Ascites Tumor (EAT) cells. Treatment with curcumin in vivo resulted in inhibition of proliferation of EAT cells and ascites formation. Further, we demonstrate that the induction of apoptosis in EAT cells showed nuclear condensation, DNA fragmentation and translocation of caspase-activated DNase (CAD) to nucleus upon curcumin treatment. Curcumin-induced apoptosis is mediated through activation of caspase-3, which is specifically inhibited by the caspase-3 inhibitor, Ac-DEVD-CHO. On the other hand, the decreased secretion of ascites by EAT cells is corroborated by reduction in VEGF secretion upon curcumin treatment. Further, CD31 immunohistological staining of peritoneum sections in curcumin-treated mice suggests its efficacy in acting as anti-angiogenic compound in EAT cells by inhibiting proliferation of endothelial cells in mouse peritoneum. However, immunoflurescence studies of NF-kB revealed that the inhibition of nuclear translocation of NF-kB p65, a transcription factor required for VEGF gene expression, in curcumin-treated EAT cells. These results suggest a further possible clinical application of this diet-derived compound curcumin, as both proapoptotic and anti-angiogenic compound in association with conventional chemotherapeutic agents.

Enhancement of the tumour inhibitory activity, in vivo, of diospyrin, a plant-derived quinonoid, through liposomal encapsulation

Diospyrin, a bisnaphthoquinonoid plant product, shows inhibitory activity against murine tumour in vivo and human cancer cell lines in vitro. Efforts have further been made to obtain synthetic derivatives of diospyrin with the objective of improved therapeutic effects. With the goal to reduce the toxicity towards normal cells and enhance the efficacy to tumour cells, diospyrin was encapsulated in liposomal vesicle and its antitumour potential was observed on the growth of Ehrlich ascites tumour in Swiss mice. It was found that the longevity of the tumour-bearing mice was significantly enhanced by treatment with liposomal diospyrin as compared with the free drug. Biochemical assay of liver function enzymes, viz. LDH, AP, GOT and GPT in blood serum of the tumour-bearing mice showed substantial alterations in the activity of these enzymes. These parameters were, however, restored to near normal level when the drug treatment was given encapsulated in a liposome. Histopathological studies on the liver tissues indicated a near normal pathological status in the treated animals despite being challenged by tumour cells. This study on diospyrin has shown, for the first time, an enhancement of its antitumour effect in vivo through liposomal encapsulation.

Depolymerized products of chitosan as potent inhibitors of tumor-induced angiogenesis

Water-soluble low-molecular weight chitosan (LMWC) and chitooligosaccharides (COs) were obtained from chitosan (16% N-acetylation) by depolymerization induced by potassium persulfate under nitrogen atmosphere for 2 h. They were characterized by IR, X-ray, HPLC and (13)C-NMR. Splitting of C3/C5 signals in the latter indicated a newer conformation, and also showed prominence of acetyl groups in LMWC, may be due to cleavage between two consecutive deacetylated residues. Molecular weight of LMWC, determined by HPSEC, showed a single peak of approximately 37 kDa. HPLC analysis of the solvent-extracted fraction revealed COs enriched with pentamer, hexamer and higher oligomers. The effect of LMWC and COs on the growth of Ehrlich ascites tumor (EAT) cells and tumor-induced neovascularization was studied. COs (50 microg) were more effective compared to LMWC (100 microg) and proved to be potent angioinhibitory and antitumor compounds, as shown by inhibition of angiogenesis and inducing apoptosis as a function of DNA fragmentation.

Dual role of polymorphonuclear neutrophils on the growth of Ehrlich ascites tumor (EAT) in mice

We show that granulocytes (PMN) have a dual role in the development of Ehrlich Ascites Tumor (EAT) in mice. EAT intraperitoneal inoculation causes a local inflammatory reaction, ascites development and mortality that distinguish resistant and susceptible strains. In resistant mice (CAF1), there is a less pronounced PMN influx after EAT inoculation than in susceptible Swiss mice. Accordingly, the increase in peritoneal PMN numbers enhanced tumor growth in CAF1 mice, but had no effect in the susceptible Swiss animals. Contrastingly, PMN depletion had no effect in resistant mice but facilitated tumor growth in susceptible animals. Though no differences were noted between the strains in peritoneal cell spreading and hydrogen peroxide release after tumor inoculation, in vitro PMN cytotoxic activity against EAT was significantly higher in susceptible Swiss mice. These data indicate a paradoxical dual role for PMN against EAT: while they help control tumor development in susceptible animals, they seem to enhance tumor growth in resistant mice.

Subpopulations of mononuclear leukocytes associated with inhibition of Ehrlich ascites tumor growth by treatment with Bothrops jararaca venom.

Snake venoms have been used as antineoplastic substances in several experimental models. We demonstrated in previous studies that Bothrops jararaca venom (BjV) induces inhibition of Ehrlich ascites tumor (EAT) growth accompanied by an increase of mononuclear (MN) leukocytes in all groups inoculated with EAT and/or venom. The objective of the present study was to characterize the subpopulations of MN leukocytes involved in the inhibition of EAT growth by treatment with BjV. Swiss mice were inoculated with 1.0x10(3) EAT cells by the intraperitoneal route and treated with 0.4 mg/kg of BjV by the same route (Group TV). Treatment was started 24 h after tumor cell inoculation and consisted of five intraperitoneal injections performed at 72 h intervals. After 2, 8 and 14 days, groups of animals were sacrificed and the number of B, TCD4 and TCD8 lymphocytes, macrophages and natural killer cells present in the peritoneal cavity was determined by flow cytometry. The control group consisted of animals inoculated with EAT and treated with 0.1 ml of saline under the same conditions as the experimental group (Group T). Two additional control groups consisted of animals not inoculated with EAT and treated with saline or venom. Data were analyzed statistically by the Kruskal-Wallis non-parametric test for independent samples. On the 2nd and 8th day we observed a difference between groups T and TV (group T > group TV) for all cell types, except natural killer cells, that only differed on the 2nd day. However, on the 14th day there was no difference in MN cells among groups. These data suggest that the inhibition of EAT is related to the toxic action of BjV on tumor cells and/or to the proteolytic effect of the venom on the mediators produced by the cells for growth modulation.

Natural Killer Cell Activity, Lymphocyte Proliferation, and Cytokine Profile in Tumor‐Bearing Mice Treated with MAPA, a Magnesium Aggregated Polymer from Aspergillus oryzae

The present study examined the effects of MAPA, an antitumor aggregated polymer of protein magnesium ammonium phospholinoleate‐palmitoleate anhydride, isolated from Aspergillus oryzae, on concanavalin A (Con A)‐induced spleen cell proliferation, cytokine production and on natural killer (NK) cell activity in Ehrlich ascites tumor‐bearing mice. The Ehrlich ascites tumor (EAT) growth led to diminished mitogen‐induced expansion of spleen cell populations and total NK activity. This was accompanied by striking spleen enlargement, with a marked increase in total cell counts. Moreover, a substantial enhancement in IL‐10 levels, paralleled by a significant decrease in IL‐2 was observed, while production of IL‐4 and interferon‐γ (IFN‐γ) was not altered. Treatment of mice with 5 mg/kg MAPA for 7 days promoted spleen cell proliferation, IL‐2 production and NK cell activity regardless of tumor outgrowth. In addition, MAPA treatment markedly enhanced IFN‐γ levels and reduced IL‐10 production relative to EAT mice. A 35% reduction in splenomegaly with normal number of nucleated cells was also found. Altogether, our results suggest that MAPA directly and/or indirectly modulates immune cell activity, and probably disengages tumor‐induced suppression of these responses. Clearly, MAPA has an impact and may delay tumor outgrowth through immunotherapeutic mechanisms.

Changes in endogenous tissue glutathione level in relation to murine ascites tumor growth and the anticancer activity of cisplatin.

Changes in glutathione levels were determined in tissues of 11- to 12-week-old Swiss albino mice at different stages of Dalton's lymphoma tumor growth and following cisplatin (8 mg/kg body weight, ip) treatment for 24-96 h, keeping 4-5 animals in each experimental group. Glutathione levels increased in spleen of tumor-bearing compared to normal mice (9.95 +/- 0.14 vs 7.86 +/- 1.64 micromol/g wet weight, P<or=0.05) but decreased in blood (0.64 +/- 0.10 vs 0.85 +/- 0.09 mg/ml) and testes (9.28 +/- 0.15 vs 10.16 +/- 0.28 micromol/g wet weight, P<or=0.05). Dalton's lymphoma cells showed an increase in glutathione concentration (4.43 +/- 0.26 micromol/g wet weight) as compared to splenocytes, their normal counterpart (3.62 +/- 0.41 micromol/g wet weight). With the progression of tumor in mice, glutathione levels decreased significantly in testes (approximately 10%) and bone marrow cells (approximately 13%) while they increased in Dalton's lymphoma cells (28-46%) and spleen (15-27%). Glutathione levels in kidney, Dalton's lymphoma cells and bone marrow cells (8.50 +/- 1.22, 4.43 +/- 0.26 and 3.28 +/- 0.17 micromol/g wet weight, respectively) decreased significantly (6.04 +/- 0.42, 3.51 +/- 0.32 and 2.17 +/- 0.14 micro mol/g wet weight, P<or=0.05) after in vivo cisplatin treatment for 24 h. Along with a decrease in glutathione level, the glutathione-S-transferase (GST) activity also decreased by 60% in tumor cells after cisplatin treatment. The elevated drug uptake by the tumor cells under the conditions of reduced glutathione concentration and GST activity after treatment could be an important contributory factor to cisplatin's anticancer activity leading to tumor regression. Furthermore, lower doses of cisplatin in combination with buthionine sulfoximine (an inhibitor of glutathione synthesis) may be useful in cancer chemotherapy with decreased toxicity in the host.

Therapeutic effects of viral vector-mediated antiangiogenic gene transfer in malignant ascites.

Malignant ascites is a common complication of advanced intraabdominal neoplasms for which standard treatments are suboptimal. Evidence suggests that tumor-mediated angiogenesis and enhanced vascular permeability in the peritoneal wall due to high levels of vascular endothelial growth factor play a fundamental role in the pathogenesis of malignant ascites. To explore the advantage of viral vector-mediated "targeted antiangiogenic therapy" in ascites formation, we constructed and administered adenoviral vectors encoding several different antiangiogenic proteins (angiostatin, endostatin, platelet factor 4, and a fusion protein between angiostatin and endostatin) alone or in combination intraperitoneally in mice with peritoneal carcinomatosis from breast cancer (TA3 cells) and ovarian cancer (SKOV-3 i.p. and ES-2 cell lines) to explore the potential of additive or synergistic activity. Our data demonstrated statistically significant downregulation of ascites formation, tumor growth, vascularity, and prolongation of animal survival after intraperitoneal treatment with antiangiogenic adenoviral vectors in three different ascites tumor models. Combined treatment proved to be more effective than treatment with one vector alone. Reduced ascites formation was accompanied by decreased microvascular density in the peritoneal wall and increased apoptosis of tumor cells after administration of antiangiogenic vectors in vivo. Of interest was the observation that AdPF4 caused a significant decrease in the level of VEGF secreted by tumor cells both in vitro and in TA3 ascites tumor-bearing animals in vivo. These data suggest that adenoviral vector-mediated delivery of genes encoding antiangiogenic proteins may represent a potentially new treatment modality for malignant ascites.

On the Inhibitory Activities of a New Boron Compound and Ultrasound against the Mouse Ascites Tumour

The inhibitory effects of a new boron compound, dihydroxy (oxybiguanido) boron (III) hydrochloride monohydrate (HB), and ultrasound (US) of a frequency 25 kHz on the growth of ascites tumour in female Swiss mice were studied by monitoring the survival, weight of tumour-associated material, tumour cell count, serum alkaline phosphatase activity and the haematological parameters of the treated animals. 5-Fluorouracil (5-FU), a well-known anticancer agent, was used as positive control. While HB exhibiited a very significant antitumour action, US alone produced a small but significant inhibitory effect. The combination of US with HB or 5-FU produced an extra antitumour action as compared to the actions of these chemicals used singly. The mechanisms of action of the new boron compound (HB) and US are discussed.

Involvement of VEGF and its receptors in ascites tumor formation.

Vascular endothelial growth factor (VEGF) has potent endothelial cell mitotic and vascular permeability activity. Several reports have suggested that VEGF may be one of the major factors regulating ascites formation, although no quantitative and systematic analyses have been carried out. To determine the role of VEGF in ascites formation, we examined the expression of VEGF in 13 mouse ascites tumors (5 sarcomas, 3 carcinomas, and 5 hematopoietic malignancies). We found that significant amounts (6-850 ng/mL) of biologically active VEGF accumulated in the ascites fluid of all 13 tumors, particularly in tumors of sarcoma and carcinoma origin (430 +/- 234 ng/mL). The microvessel densities in the peritoneal walls of tumor-bearing mice, which are significantly higher than those in healthy mice, basically correlated with but did not parallel VEGF concentrations, suggesting the existence of an additional modulator(s) of the angiogenic process. Administration of anti-mouse VEGF-neutralizing antibody to mice bearing the carcinoma-derived ascites tumor MM2 suppressed ascites accumulation, tumor growth, and tendency to bleed. These results directly demonstrate the crucial role of VEGF in carcinoma-derived ascites tumor formation in vivo.

Effect of the seed extract of carrot (Daucus carota Linn.) on the growth of Ehrlich ascites tumour in mice

The petroleum ether extract of the seeds of carrot (Daucus carota Linn), administered intraperitoneally at doses of 3 mg and 10 mg/kg body weight/day for 7 days, showed antitumour activity, inhibiting the growth of Ehrlich ascites tumour in mice. Copyright © 1998 John Wiley & Sons, Ltd.