Effect of combined administration of a synthetic low-toxicity lipid A derivative,DT-5461a,and indomethacin in various experimental tumor models of colon 26 carcinoma in mice

We investigated the antitumor effects of a synthetic lipid A derivative, DT-5461a, in combination with indomethacin in three experimental tumor models (peritoneal carcinomatosis, liver tumor, and lung tumor models) of transplanted colon 26 carcinoma in mice. This carcinoma produces the immunosuppressive prostaglandin E₂ (PGE₂). Intravenous administration of DT-5461a alone resulted in little or no prolongation of survival time [increase in life span (ILS): –2%–22%]. When indomethacin was given in drinking water a slight or moderate increase in survival time was seen (ILS: 4%–45%). In contrast, the combination of DT-5461a and indomethacin induced an additive increase in life span (ILS: 16% to more than 193%). The strongest antitumor effect of this combined therapy was seen in the peritoneal carcinomatosis model; in this model, plasma PGE₂ concentrations were considerably higher than in normal mice, and concentrations were further but transiently increased by DT-5461a administration. Following oral indomethacin administration, these elevated PGE₂ concentrations were reduced to the level in untreated normal mice. Furthermore, intratumoral tumor necrosis factor (TNF) activity in the group receiving the combined therapy was significantly higher than that in the DT-5461a-treated group. No TNF production was induced by the administration of indomethacin alone. These results suggest that the antitumor effect of DT-5461a can be enhanced by combination with indomethacin, and that the inhibition of PGE₂ production may have a role in this antitumor effect.This study is dedicated to the memory of the late Dr. Yasuaki Osada, in acknowledgement of his encouragement and support     

Synergistic antitumor activity of interleukin-2 and cimetidine against syngeneic murine tumor

Summary Cimetidine, an H2 histamine receptor antagonist, is a potent immunomodulating agent, which acts by inhibiting suppressor T lymphocyte function. The present work investigated the effect, if any, of cimetidine on interleukin-2 (IL-2)-induced natural killer (NK) and lymphokine-activated killer (LAK) cell activities, and on in vivo antitumor activity using syngeneic colon 26 adenocarcinoma as the model. Mimicking the clinical conditions, all in vitro experiments were evaluated with the splenocytes prepared from tumor-bearing BALB/c mice. Ten days after subcutaneous inoculation of tumor cells (5 × 10⁵), animals were treated intraperitoneally daily with phosphate-buffered saline (PBS), cimetidine (2 mg kg^–1 day^–1), IL-2 (300 000 IU/day), or cimetidine plus IL-2 for 7 consecutive days. The treatment of IL-2 plus cimetidine increased NK and LAK cell activities significantly and synergistically at the end of the treatment (i.e. on day 18) as well as 1 week after the treatment (i.e. on day 25), in comparison with those of the control groups (PBS, cimetidine alone, IL-2 alone). Also, in vivo antitumor activity, as analyzed by a Kaplan-Meier life table with the log-rank test, revealed a significantly prolonged survival in the group treated with IL-2 plus cimetidine compared to the control groups. Phenotyping performed on the murine splenocytes on day 18 indicated a significant reduction in Lyt2-positive cells in the cimetidine-treated group in comparison with the PBS group. A significant increase in asialo GM1-positive cells and IL-2-receptor-positive cells was detected in the group treated with IL-2 plus cimetidine in comparison with the PBS and IL-2 control groups. Therefore, this study indicates a synergistic enhancement of IL-2-induced NK and LAK cell activities in tumor-bearing hosts by cimetidine, a noncytotoxic inhibitor of suppressor T function, and a significantly prolonged survival of tumor-bearing animals treated by IL-2 plus cimetidine. It also suggests the clinical potential of combination therapy of IL-2 with cimetidine.     

Activation of tumor-infiltrating macrophages by a synthetic lipid A analog (ONO-4007) and its implication in antitumor effects

ONO-4007 is a novel synthetic analog of lipid A subunit and has been shown to exert antitumor activities on various experimental tumors with less toxicity than lipopolysaccharide. It remains unclear, however, what biological activities of this compound are relevant to its antitumor effects. We therefore investigated the activation of macrophages by ONO-4007 in vitro and in vivo and its implication in antitumor effects, using mouse MM46 mammary tumor as an experimental model. Intravenous injection of ONO-4007 produced significant therapeutic effects on this solid tumor. ONO-4007 could stimulate glycogen-elicited peritoneal macrophages in vitro, not only to produce tumor necrosis factor (TNF), but also to exert cytocidal activities against MM46 cells in vitro. Substantial TNF production was induced in tumor tissue by i. v. injection of ONO-4007, and its successive administration to tumor-bearing mice gave tumor-infiltrating macrophages a prominent in vitro tumoricidal activity and primed them for in vitro TNF secretion. These results suggest that activation of tumor-infiltrating macrophages to a direct tumoricidal state as well as to TNF secretion in tumor tissues may be at least some of the antitumor effects of this novel lipid A analog.     

A MurG assay which utilises a synthetic analogue of lipid I

A standard assay for the MurG enzyme using a lipid I analogue [MurNAc(N(epsilon)-dansylpentapeptide)-pyrophosphoryl (R,S)-alpha-dihydroheptaprenol] and radioactive UDP-N-acetylglucosamine was set up. A high concentration (35%) of dimethylsulfoxide was necessary for maximal activity. Separation and quantitation were accomplished by reverse-phase high performance liquid chromatography (HPLC) in isocratic conditions and on-line radioactivity detection, thereby providing a rapid and accurate assay. The kinetic parameters of the MurG reaction were determined; the reaction was shown to also catalyse the reverse reaction at a measurable rate. A lipid I analogue containing dihydroundecaprenol as the prenyl chain turned out to be a poor MurG substrate, presumably owing to aggregation.     

Therapeutic effects of a synthetic peptide of C-reactive protein in pre-clinical tumor models

Previous studies have shown that multilamellar vesicles (MLV) or other carriers containing purified human C-reactive protein (CRP) have therapeutic activity in preclinical tumor models. Here we evaluated the therapeutic effects of MLV containing novel synthetic peptides, derived from the structure of CRP, on the extent of (a) established lung metastases of fibrosarcoma T241 in C57B1/6 mice, (b) survival of C57B1/6 mice bearing established liver metastases of colon carcinoma MCA-38, and (c) primary tumor growth of Renca renal carcinoma in Balb/c mice. In all cases, a single synthetic CRP peptide, RS-83277, demonstrated significant antitumor effects comparable to that seen with intact CRP. Two other synthetic CRP peptides, RS-83287 and RS-83147, showed no therapeutic activity and were comparable to control MLV containing only buffer. None of the peptides contained sequences homologous with that of the phagocyte stimulant, tuftsin. Activity of MLV-encapsulated RS-83277 was dose-dependent, and a comparable dose of the soluble peptide, given either alone or following injection of buffer-MLV, was ineffective. These results demonstrate immunotherapeutic potential for a novel synthetic peptide derived from CRP, and endogenous acute-phase protein.This work was supported in part by grant CA 49950 from the National Cancer Institute, and grant 43618 from the National American Cancer Society     

Analysis of effector T cells against the murine syngeneic tumor MethA in mice orally administered antitumor polysaccharide SPR-901

The growth of MethA tumor was significantly inhibited by oral administration of the -glucan SPR-901 in BALB/c (+/+) mice but not in nude mice. Mice treated orally with SPR-901 exhibited an augmentation of antigen-specific resistance against rechallenge with the tumor cells. The tumor-neutralizing activity of regional lymph node cells from MethA-bearing mice against the tumor was augmented by oral administration of SPR-901. The tumor-neutralizing activity of lymph node cells from SPR-901-treated mice mainly appeared in Lyt2+cells. Furthermore, lymphokine-activated killer activity of these cells was enhanced by administration of SPR-901. The antitumor effect of SPR-901 was abrogated in mice depleted of either L3T4+ or Lyt2+ cells, and in cyclosporin-A-treated mice. These results suggest that Lyt2+ cells are important effector cells in MethA-bearing mice orally adminstered SPR-901 and that functional exertion of both Lyt2+ and L3T4+T cells is necessary for the antitumor effect of orally administered SPR-901 in vivo.     

Modulation of lipopolysaccharide-induced production of tumor necrosis factor, interleukin 1, and interleukin 6 by synthetic precursor Ia of lipid A

Abstract Endotoxin (lipopolysaccharide, LPS) induces the production of mediators of inflammation, which exerts pathophysiological effects such as fever or shock in mammals. In the present study we have investigated the modulation of LPS by the synthetic non-active tetraacylated precursor Ia of lipid A (compound 406) in the induction of tumor necrosis factor (TNF), interleukin 1 (IL-1) and interleukin 6 (IL-6) in human peripheral blood mononuclear cells (PBMC) and in human peripheral blood monocytes (PBMo). PBMC stimulated with LPS released TNF in a concentration dependent manner. Release of biologically active TNF, IL-1 and IL-6 was first detectable 4 h after LPS stimulation. Compound 406 alone in all concentrations tested did not induce TNF, IL-1 or IL-6 release, intracellular TNF or IL-1β, or mRNA for TNF or IL-1. Added to PBMC 1 h before LPS compound 406 enhanced or suppressed TNF release and suppressed IL-1 and IL-6 release depending on the ratio of concentrations between stimulator (LPS) and modulator (compound 406). In contrast to LPS stimulation alone TNF, IL-1 and IL-6 release in presence of compound 406 was delayed and first detectable after 6 to 8 h. Compound 406 was able to suppress LPS-induced intracellular TNF and IL-1β in PBMC. Added to PBMo 1 h before LPS it totally inhibited the production of mRNA for TNF and IL-1. When added to PBMC 1 h after LPS, TNF release was suppressed in a concentration-dependent way and release of biologically active TNF, IL-1 and IL-6 could again be detected for the first time after 4 h. Compound 406 was not able to inhibit phorbol 12-myristate 13-acetate (PMA)-induced TNF and IL-1 release in PBMo which suggests that its modulating effect is LPS-specific. This study provides evidence that the modulating effect of compound 406 on the LPS induction of TNF, IL-, 1 and IL-6 could be due to competitive binding.     

In vivo antitumor effects of murine interferon- γ -inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites

Interferon-γ-inducing factor/interleukin-18 is a novel cytokine that reportedly augments natural killer (NK) activity in human and mouse peripheral blood mononuclear cell cultures in vitro and has recently been designated IL-18. In this study, IL-18 exhibited significant antitumor effects in BALB/c mice challenged intraperitoneally (i.p.) with syngeneic Meth A sarcoma when administered i.p. on days 1, 2 and 3 after challenge. Intravenous (i.v.) administration also induced antitumor effects in the tumor-bearing mice; however, subcutaneous (s.c.) administration did not. When mice were twice pretreated with 1 μg IL-18 3 days and 6 h before tumor challenge, all mice survived whereas control mice died within 3 weeks of challenge. Inhibitory effects on Meth A cell growth in vitro were not observed with either IL-18 or interferon γ. The effects of IL-18 pretreatment were abrogated by abolition of NK activity after mice had been injected with anti-asialo GM1 antibody 48 h before and, 24 h and 72 h after tumor challenge. Mice pretreated with IL-18 and surviving tumor challenge resisted rechallenge with Meth A cells but could not reject Ehrlich ascites carcinoma, and spleen cells from the resistant mice, but not control mice, exhibited cytotoxic activity against Meth A cells in vitro after restimulation with mitomycin C-treated Meth A cells for 5 days. The effector cells in the spleen cell preparations from resistant mice appear to be CD4⁺ cells because cytolytic activity was significantly inhibited after depletion of this subset by monoclonal antibodies and complement. In conclusion, IL-18 exhibits in vivo immunologically (primarily NK) mediated antitumor effects in mice challenged with syngeneic Meth A sarcoma and induces immunological memory and the generation of cytotoxic CD4⁺ cells. Received: 17 September 1996 / Accepted: 8 November 1996     

Synthesis of a dimeric monosaccharide lipid A mimic and its synergistic effect on the immunostimulatory activity of lipopolysaccharide

Bacterial endotoxin lipopolysaccharide (LPS) is a potent immune stimulant, with the recognition of LPS and its active principal lipid A mediated by the Toll-like receptor 4 (TLR4)/MD-2 receptor complex. Due to the broad downstream implications of TLR4-mediated signalling, TLR4 ligands show great potential for immunotherapeutic manipulations. In this paper a dimeric monosaccharide lipid A mimic (3) has been designed as a potential TLR4 ligand. The chemical synthesis and the preliminary biological studies are described. Compound 3 shows a significant synergistic effect on LPS-induced ICAM-1 expression in human monocytic THP-1 cells.     

Augmentation of antitumor effect on syngeneic murine solid tumors by an interleukin 2 slow delivery system,the IL-2 mini-pellet

We evaluated the antitumor effect of an interleukin 2 (IL-2) slow delivery system, the IL-2 mini-pellet, in two murine solid tumor models, and also investigated the enhancement of its therapeutic effect by serial administration. The IL-2 mini-pellet contains 1 × 106 units of IL-2 and releases it slowlyin vivo. In our experiment, the IL-2 mini-pellet was administered subcutaneously near the tumor site in combination with the intravenous injection of lymphokine-activated killer (LAK) cells. When this regimen was given on days 8 and 11 after the subcutaneous inoculation of Meth A fibrosarcoma into BALB/c mice, tumor growth was significantly inhibited (p < 0.05) compared to tumor growth in untreated controls. Moreover, the IL-2 mini-pellet alone was also effective in inhibiting tumor growth. In another experiment, MH134 hepatoma was inoculated into C3H/He mice. Both administration of the IL-2 minipellet alone and in combination with LAK cells resulted in complete tumor regression in four of five mice. In a third experiment, serial administration of the IL-2 mini-pellet at 3- or 5-day intervals prolonged the suppression of Meth A fibrosarcoma growth in BALB/c mice. These results suggested that the IL-2 mini-pellet could be applied to cancer immunotherapy and that its antitumor effect could be prolonged by serial administration.     

Suppressive effect of lipid A partial structures on lipopolysaccharide or lipid A-induced release of interleukin 1 by human monocytes

Abstract Experiments were designed to investigate the significance of lipid A partial structures, precursor Ia (compound 406), and lipid X (compound 401) to serve as antagonists of interleukin 1 (IL-1) release from human mononuclear cells and monocytes induced by lipopolysaccharide (LPS, endotoxin) of Salmonella aborus equi or synthetic Escherichia coli lipid A (compound 506). A definite inhibition mediated by lipid A partial structures on IL-1 release induced by LPS or lipid A was found in repeated experiments. The inhibitory effect was exterted not only on IL-1 release, but also on IL-1 peptide synthesis at the intracellular level. The results also show that lipid A partial structures have suppressive effects even when added 1–4 after LPS or lipid A. We conclude from these results that lipis A partial structures (precursor Ia and lipid X) have potent immunomodulatory effects on LPS- and lipid A-induced IL-1 release and may become useful reagents to study the mechanism of interaction of LPS and lipid A with cells of the immune system.     

Diphosphoryl lipid A derived from the lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 is a potent competitive LPS inhibitor in murine macrophage-like J774.1 cells

Abstract Pentaacyl diphosphoryllipid A derived from the nontoxic lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 (RsDPLA) did not induce tumour necrosis factor-α nor interleukin-6 release in the murine macrophage-like cell line J774.1. However, it effectively inhibited the induction of these two cytokines by LPS of Salmonella minnesota Re mutant R595 (ReLPA) in a concentration-dependent manner. Maximal inhibition and half-maximal inhibition occured when the ReLPS to RsDPLA mass ratio was 1:30 and 1:1, respectively. A binding study was performed in the presence of serum to determine whether RsDPLA is competing with ReLPS for LPS binding sites on J774.1 cells. This assay allows the determination of LPS binding to J774.1 cells via a mechanism involving CD14, a receptor for complexes of LPS with LPS binding protein (LBP), and its possible inhibition. The results show that RsDPLA strongly inhibits the binding of ¹²⁵I-labelled ReLPS to J774.1 cells. Maximal and one-half maximal inhibition of binding occured when the ReLPS to RsDPLA mass ratios were 1:2.5 and 1:0.5, respectively. It was found that the inhibition of binding by RsDPLA was much stronger than that by unlabelled ReLPS. These results suggest that RsDPLA is competing with ReLPS for CD14-dependent recognition of LPS on J774.1 cells.     

Augmentation of antitumor effect by combined administration with interleukin-2 and sizofiran,a single glucan,on murine EL-4 lymphoma

The antitumor effect of the combined administration with recombinant human interleukin-2 (rIL-2) and sizofiran (SPG), a single glucan of Shizophyllum commune Fries, was studied in vivo in C57BL/6 mice intraperitoneally inoculated with EL-4 lymphoma. The effect was evaluated by a) comparing the survival time of the mice, b) analysis of the intraperitoneal cell population in Giemsa-stained specimens, c) surface marker analysis of peritoneal exudative cells with flow cytometry, d) cytotoxic assay of cells against EL-4 and Yac-1 lymphoma, and e) elimination of some cell populations by monoclonal antibodies, to identify the antitumor-effector cells showing cytotoxic activity. The survival of mice given both rIL-2 and SPG was significantly longer than the control mice or those given SPG alone or rIL-2 alone. It was demonstrated that the administration of SPG and/or rIL-2 to the EL-4 lymphoma-bearing mice activated immune-response cells in the peritoneal cavity such as T lymphocytes, NK cells, or macrophages, which might be effective in reducing lymphoma cells. The combination of rIL-2 and SPG administration appears to activate the antitumor- immune response at the tumor site more effectively than when either agent was administered alone.     

Antiallergic effect of ardisiaquinone A, a potent 5-lipoxygenase inhibitor

The antiallergic effects of ardisiaquinone A, a potent 5-lipoxygenase inhibitor, were examined. Pretreatment with ardisiaquinone A (0.1–10 μM) significantly inhibited compound 48/80-induced production of cysteinyl-leukotrienes (cys-LTs; LTC₄, LTD₄ and LTE₄) in rat peritoneal mast cells, but not histamine release. The IC₅₀ value was 5.56 μM. Pre-administration with ardisiaquinone A (0.1–1 mg/kg, s.c.) dose-dependently inhibited rat homologous passive cutaneous anaphylaxis (PCA) and the maximal inhibitory ratio was 22.3 ± 3.9% at the dose of 1 mg/kg. Ardisiaquinone A (1–5 mg/kg, s.c.) dose-dependently prevented the allergen-induced increase of tracheal pressure in ovalbumin-sensitized guinea pigs, especially during the late phase.

In conclusion, the findings of this study show that 5-lipoxygenase inhibitor ardisiaquinone A partially attenuates the allergen-induced increases of vascular permeability and tracheal pressure via the inhibition of cys-LTs production in mast cells.     

Induction of intratumoral tumor necrosis factor by a synthetic lipid A analog, ONO-4007, with less tolerance in repeated administration and its implication in potent antitumor effects with low toxicity

To evaluate the anti-tumor characteristics of ONO-4007, a synthetic analog of lipid A, the authors examined its acute toxicity and anti-tumor activity in a mouse MM46 mammary tumor system in comparison with LA-15-PP, an E. coli-type synthetic lipid A and LPS. Systemic and local (tumor site) induction of tumor necrosis factor (TNF) by a single i.v. shot of ONO-4007 and LA-15-PP correlated with manifestation of their toxicity, showing that ONO-4007 is 100-fold less effective than LA-15-PP. However, a protocol of repeated administration (3 shots twice a week) exhibited about 10 times more therapeutic potency of ONO-4007 for cancer therapy than expected in the above experiments. In a dose inducing submaximal systemic and intratumoral TNF production, repeated injections (twice a week) of ONO-4007 (10 mg/kg), LA-15-PP (0.1 mg/kg) and LPS (0.1 mg/kg) commonly generated a tolerant state in the systemic response (serum and liver) to subsequent stimulation. The intratumoral response was retained with this repeated administration of ONO-4007, but was not with LA-15-PP or LPS. TIM (tumor-infiltrating macrophages) isolated from mice pre-injected with ONO-4007 and LA-15-PP were found to lose their response to both substances, but the response was rapidly recovered until 72 h after injection and virtually no difference was observed in their response to either drug. The in vitro treatment of naive TIM with ONO-4007 or LA-15-PP for 2 h depressed the response to both substances and the depression continued for 72 h even in culture with fresh medium. The relatively high efficacy of ONO-4007 in cancer therapy likely depends on the retraction of the tolerant state, especially at the tumor site where the response to ONO-4007 is recovered much more efficiently than that to lipid A. While constant recruitment of macrophages to tumor tissue might be involved in the difference of tolerance recovery between this region and others, selective response to ONO-4007 may not be explained simply by the sensitivity of recruited TIM. Pharmacokinetical experiments revealed that repeated injections of LA-15-PP enhanced its clearance from blood circulation, while the clearance of ONO-4007 was stable after repeated injections. Thus, pharmacokinetical properties of ONO-4007 may also possibly be implicated in this event.     

Evaluation in humans of ELF-EMF exposure on chromogranin A,a marker of neuroendocrine tumors and stress

ABSTRACT

Chromogranin A (CgA), which is a major protein in adrenal chromaffin cells and adrenergic neurons, is a clinically relevant endocrine and neuroendocrine tumor marker including pheochromocytomas, neuroblastomas, and related neurogenic tumors. In this study, we looked at the effect in humans of chronic daily exposure to a 50-Hz magnetic field. We examined in 15 men (38.0 ± 0.9 years) the effects of chronic daily exposure to a 50-Hz magnetic field for 1–20 yrs both at home and at work. EMDEX II dosimeters were used to record magnetic field all day long every 30 s. for 1 week. The weekly geometric mean of the individual exposures ranged from 0.1 to 2.6 μT. Blood samples were taken hourly between 20:00 h and 08:00 h. CgA patterns of exposed subjects were compared to age-matched controls. The results of exposed subjects were compared with those for 15 unexposed men who served as controls and whose individual exposure was ten times lower ranging from 0.004 to 0.092 μT. This work shows that in the control group the serum CgA levels exhibited a nighttime peak with a progressive decline of the serum concentrations and a nadir in the morning. Both the profile and the serum concentrations of CgA, a marker of neuroendocrine tumors and stress, did not appear to be impaired in the subjects chronically exposed over a long period (up to 20 yrs) to magnetic fields though a trend toward lower levels were found at the highest exposure (>0.3 μT). This does not rule out, however, that the potential deleterious risk of ELF-EMF on frail populations such as children and the elderly may be greater at low exposure and should hence be documented, at least for their residential exposure.     

The effect of cytochrome oxidase on lipid chain dynamics A nanosecond fluorescence depolarization study

Molecular motions in membranes composed of purified cytochrome oxidase (EC 1.9.3.1) and synthetic lipid (l-α-dimyristoylphosphatidylcholine or l-α-dioleoylphosphatidylcholine) at various ratios were investigated with a lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Nanosecond fluorescence depolarization kinetics of the probe showed that the rod-shaped probe molecules perform a fast wobbling motion (restricted rotation) in all membranes studied, presumably reflecting the motion of lipid acyl chains. At temperatures where the pure lipid was in the liquid-crystalline phase, presence of cytochrome oxidase reduced the angular range of the wobbling motion, whereas its rate; the wobbling diffusion constant, was unaffected. On the other hand, incorporation of the protein into lipid in the gel phase resulted in the increase in the wobbling diffusion constant while the range of the wobbling motion remained the same. A time-dependent view of lipid dynamics that accounts for the above findings, as well as the results of recent electron spin resonance and nuclear spin resonance studies of protein-lipid interactions, is proposed.