lipid-a and Colonic-Neoplasms

A vaccine consisting of four allogeneic colon carcinoma cell lines (DLD-1, HCT116, WiDr, and T84) mixed with the adjuvant DETOX (Mycobacterium phlei cell wall and Salmonella minnesota lipid A) was administered to 25 patients with low-volume metastatic colorectal carcinoma. The first eight patients received vaccine only, given intradermally on three occasions at 3-week intervals. Subsequent patients also received subcutaneous interleukin-1 alpha (IL-1 alpha), 0.3-0.5 microgram/m2 per day for 8 days after each vaccination in an outpatient setting. Vaccine alone caused local erythema, induration, and pruritus. IL-1 caused fevers, chills, and rigors that started in 4 h and lasted 1-2 h. One patient developed a brief loss of consciousness with a rigor that resolved without sequelae. One episode of mild hypotension occurred. Fatigue occurred by day 8 of IL-1. A substantial increase in the number of patients with positive skin tests to DLD-1 and HCT116 occurred after vaccine treatment both without and with IL-1 alpha. An allogeneic cell vaccine plus subcutaneous IL-1 was administered safely to outpatients with some evidence of in vivo effect observed.

Topics: Adjuvants, Immunologic; Adult; Aged; Cancer Vaccines; Colonic Neoplasms; Female; Hepatitis B Antibodies; Humans; Immunotherapy, Active; Interleukin-1; Lipid A; Male; Middle Aged; Mycobacterium; Neoplasm Metastasis; Rectal Neoplasms; Skin Tests; Treatment Outcome

Fas belongs to the family of tumor necrosis factor receptors which induce apoptosis. Many cancer cells express Fas but do not undergo Fas-mediated apoptosis. Nitric oxide reverses this resistance by increasing levels of Fas at the plasma membrane. We studied the mechanisms by which NO affects Fas function.. Colon and mammary cancer cell lines were incubated with the NO donor glyceryl trinitrate or lipid A; S-nitrosylation of Fas was monitored using the biotin switch assay. Fas constructs that contained mutations at cysteine residues that prevent S-nitrosylation were used to investigate the involvement of S-nitrosylation in Fas-mediated cell death. Apoptosis was monitored according to morphologic criteria.. NO induced S-nitrosylation of cysteine residues 199 and 304 in the cytoplasmic part of Fas. In cancer cells that overexpressed wild-type Fas, S-nitrosylation induced Fas recruitment to lipid rafts and sensitized the cells to Fas ligand. In cells that expressed a mutant form of Fas in which cysteine 304 was replaced by valine residue, NO-mediated translocation of Fas to lipid rafts was affected and the death-inducing signal complex and synergistic effect of glyceryl trinitrate-Fas ligand were inhibited significantly. These effects were not observed in cells that expressed Fas with a mutation at cysteine 199.. We identified post-translational modifications (S-nitrosylation of cysteine residues 199 and 304) in the cytoplasmic domain of Fas. S-nitrosylation at cysteine 304 promotes redistribution of Fas to lipid rafts, formation of the death-inducing signal complex, and induction of cell death.

Topics: Animals; Apoptosis; Biotinylation; Cell Line, Tumor; Colonic Neoplasms; Cysteine; Fas Ligand Protein; fas Receptor; Female; Humans; Lipid A; Mammary Neoplasms, Experimental; Membrane Microdomains; Mice; Mutation; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Protein Processing, Post-Translational; Protein Transport; Signal Transduction; Time Factors; Transfection

Blackberries are rich in polyphenols, including anthocyanins. Polyphenols are hypothesized to have biological activities that may impact positively on human health. In these studies, an anthocyanin-rich extract from Hull blackberries grown in Kentucky was obtained and fully characterized in terms of total anthocyanin and phenolic content, polymeric color, anthocyanin composition, and total antioxidant capacity. In vitro cell culture studies showed that the blackberry extract inhibited HT-29 colon tumor cell growth in a concentration-dependent manner with 49.2 microg of total anthocyanins/mL inhibiting HT-29 cell growth up to 66% at 72 hours. Likewise, in a concentration-dependent manner, total anthocyanin concentrations in the range of 0-40 microg/mL suppressed both high-dose (10 microg/mL) and low-dose (0.1 microg/mL) lipid A-induced interleukin-12 release from mouse bone marrow-derived dendritic cells. These results suggest that Hull blackberry extract (HBE) has potent antioxidant, antiproliferative, and anti-inflammatory activities and that HBE-formulated products may have the potential for the treatment and/or prevention of cancer and/or other inflammatory diseases.

Topics: Animals; Anthocyanins; Anti-Inflammatory Agents; Cell Division; Colonic Neoplasms; Dendritic Cells; Flavonoids; Fruit; HT29 Cells; Humans; Interleukin-12; Lipid A; Mice; Phenols; Plant Extracts; Polyphenols; Rosaceae

Despite the wide range of available therapies, human colon cancers remain difficult to cure. Evidence for efficient antitumoral immune responses to be raised is now widely accepted, and numerous strategies exploiting the host immune system have been developed. A treatment based on the lipid-A derivative OM-174 has been developed in our laboratory. OM-174 induces the rejection of tumors established by injection of PROb colon cancer cells in syngeneic BDIX rats. Our immunohistochemistry study demonstrated that OM-174 treatment is associated with tumor cell apoptosis. Caspase 3 activation was detected 24 h after the first OM-174 injection. Six days after the beginning of the treatment, dendritic cells were the first immune cells that invaded tumor nodules. When dendritic cells came into contact with apoptotic tumor cells, an increased expression of the costimulatory molecules B7-1 and B7-2 was detected at the surface of these cells. Five days later, macrophages were found in the tumor nodules. Lymphocytes organized into a crown surrounding the nodules that progressively regressed during the treatment. T lymphocytes were not in contact with tumor cells or apoptotic cells at any time point. The kinetics of tumor cell apoptosis induced by OM-174, as well as the appearance of innate followed by adaptative immune cells in the tumor nodules, were compatible with cell activation and the development of immune response.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Cell Division; Colonic Neoplasms; Kinetics; Lipid A; Lipopolysaccharides; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Male; Neoplasm Transplantation; Rats; Rats, Inbred Strains; Transplantation, Isogeneic

Surgical trauma inhibits immune function. Our goal was to study the effect of surgical intervention on the development of the immune response to epithelial cell adhesion molecule (EpCAM [GA-733]), a tumor-associated protein used for vaccination in colon cancer.. Recombinant GA-733 and monophosphoryl-lipid A (MPLA) were incorporated into biodegradable beads and implanted in the following groups of mice: control, insufflation, and laparotomy. After surgery, the mice were inoculated with GA-733-transfected C26 cells (C26-EpCAM). Plasma anti-GA733 IgG antibodies were detected in enzyme-linked immunoassay (ELISA). Killing specific to GA-733 was assayed by C26-EpCAM-killing assay.. The difference in tumor size between immunized and nonimmunized animals was statistically significant only in control mice (p < 0.05). Greater cytotoxic response to C26-GA733 developed in all immunized mice groups than in their respective controls. However, anti-GA733 IgG increased significantly in the control and insufflation groups, but not in the laparotomy group.. Combined GA-733 vaccine allows reduction of tumor growth in control but not in surgically managed animals. This vaccine can induce a specific-cell and antibody-mediated immune response. Open surgery leads to a decreased antibody response to the GA-733 tumor vaccine.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Neoplasm; Antigens, Neoplasm; Cancer Vaccines; Carbon Dioxide; Cell Adhesion Molecules; Colonic Neoplasms; Enzyme-Linked Immunosorbent Assay; Epithelial Cell Adhesion Molecule; Female; Immunity, Cellular; Immunization; Insufflation; Laparotomy; Lipid A; Mice; Mice, Inbred BALB C; Tumor Cells, Cultured

Whole autologous colon cancer vaccines in combination with various adjuvants have been used in both animals and humans. At this writing, vaccine regimens have been initiated in humans 3 to 6 weeks postoperatively. This delay between tumor resection and vaccination gives surviving tumor cells an opportunity to establish themselves. Vaccine administered either preoperatively or immediately after surgery, in theory, should be more effective. However, surgery-related immunosuppression may diminish the effectiveness of pre-operative or early postoperative vaccines. This problem may be overcome by limiting postoperative immunosuppression via the use of minimally invasive methods. Alternatively, the impact of the vaccine may be improved by encapsulating the vaccine, plus adjuvant, which in theory, should extend exposure time. Encapsulation of cancer vaccines in polysaccharide particles has not yet been studied. The goal of this study was to determine whether vaccine encapsulation, preoperative vaccination, and early postoperative vaccination affected the tumor burden. In addition, laparotomy and carbon dioxide insufflation were compared.. Vaccine was prepared from ultraviolet-irradiated C26 colon cancer cells in combination with monophosphoryl lipid A, either in suspension or entrapped in alginate beads. The C26 cell line and syngeneic BALB/c mice were used for all the studies. Tumor volumes were determined after excision of the tumors 2 weeks after inoculation in these studies.. Encapsulated vaccine was more effective than the standard liquid vaccine. Significantly smaller tumors were noted in mice receiving encapsulated vaccine than in either the control group (p <0.01) or the liquid vaccine group (p <0.05). The use of a preoperative encapsulated vaccine was associated with significantly smaller tumors after laparotomy, pneumoperitoneum, or anesthesia alone when the tumors were established immediately after surgery. With an already established tumor, encapsulated vaccine, when given in the early postoperative period to mice that had undergone laparotomy or anesthesia alone was associated with significantly smaller tumors that those found in control animals.. The incorporation of a whole-cell vaccine and monophosphoryl lipid A into alginate beads increases the efficacy of pre-operative and early postoperative tumor vaccines in the setting of both laparotomy and Carbon dioxide pneumoperitoneum. The use of perioperative vaccines may prove to be an effective way to immunize patients with cancer undergoing surgery.

Topics: Adenocarcinoma; Adjuvants, Immunologic; Alginates; Animals; Cancer Vaccines; Capsules; Carbon Dioxide; Colonic Neoplasms; Colorectal Neoplasms; Drug Delivery Systems; Female; Glucuronic Acid; Hexuronic Acids; Insufflation; Laparotomy; Lipid A; Mice; Mice, Inbred BALB C; Microspheres; Neoplasm Transplantation; Polysaccharides; Postoperative Care; Preoperative Care; Tumor Cells, Cultured

Results from our previous studies demonstrated that activation of Toll-like receptor 4 (Tlr4), the lipopolysaccharide (LPS) receptor, is sufficient to induce nuclear factor kappaB activation and expression of inducible cyclooxygenase (COX-2) in macrophages. Saturated fatty acids (SFAs) acylated in lipid A moiety of LPS are essential for biological activities of LPS. Thus, we determined whether these fatty acids modulate LPS-induced signaling pathways and COX-2 expression in monocyte/macrophage cells (RAW 264.7). Results show that SFAs, but not unsaturated fatty acids (UFAs), induce nuclear factor kappaB activation and expression of COX-2 and other inflammatory markers. This induction is inhibited by a dominant-negative Tlr4. UFAs inhibit COX-2 expression induced by SFAs, constitutively active Tlr4, or LPS. However, UFAs fail to inhibit COX-2 expression induced by activation of signaling components downstream of Tlr4. Together, these results suggest that both SFA-induced COX-2 expression and its inhibition by UFAs are mediated through a common signaling pathway derived from Tlr4. These results represent a novel mechanism by which fatty acids modulate signaling pathways and target gene expression. Furthermore, these results suggest a possibility that propensity of monocyte/macrophage activation is modulated through Tlr4 by different types of free fatty acids, which in turn can be altered by kinds of dietary fat consumed.

Topics: Adenocarcinoma; Animals; Base Sequence; Cell Line; Colonic Neoplasms; Consensus Sequence; Cyclooxygenase 2; Docosahexaenoic Acids; Drosophila Proteins; Enzyme Induction; Fatty Acids, Nonesterified; Fatty Acids, Unsaturated; Gene Expression Regulation, Enzymologic; Genes, Reporter; Humans; Isoenzymes; Lauric Acids; Lipid A; Lipopolysaccharides; Macrophages; Membrane Glycoproteins; Membrane Proteins; Mice; Monocytes; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Receptors, Cell Surface; Toll-Like Receptor 4; Toll-Like Receptors; Tumor Cells, Cultured

It is well documented that nitric oxide (NO) is an effector molecule of macrophage-mediated tumor cell toxicity in vitro; however, little is known about the role of NO in the antitumor immune response in vivo. We have developed a treatment protocol using lipid A. We have investigated the effects of lipid A on inducible NO synthase (NOS II) expression and evolution inside tumors during the course of treatment. Lipid A (OM-174) treatment induced tumor regression in rats bearing established colon tumors. Furthermore, NO was synthesized and secreted inside the tumors of lipid A-treated rats, as demonstrated by the increase of NOS II mRNA and NOS II content in the tumors, as well as of NOS II activity and NO production. During treatment, NOS II was localized in tumor cells only. Lipid A had no direct effect on tumor cells in vitro, while the combination of interferon gamma (IFN-gamma) plus interleukin-1 beta (IL-1beta) induced production of NO by tumor cells which was cytostatic. The content of IFN-gamma and IL-1beta in tumors was enhanced during lipid A treatment; this is in agreement with an indirect effect of lipid A in vivo via the IFN-gamma and IL-1beta pathways.

Topics: Adenocarcinoma; Animals; Cell Division; Colonic Neoplasms; Female; Interferon-gamma; Interleukin-1; Lipid A; Male; Neoplasm Transplantation; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Inbred Strains; RNA, Messenger; Tumor Cells, Cultured

Transforming growth factor-beta1 (TGF-beta1) has been shown to down-regulate NO synthesis in a variety of normal cells. In the present study, we investigated the influence of TGF-beta1 upon NO production in tumor cells and its consequences for tumor development. During the growth of PROb colon carcinoma cells intraperitoneally injected in syngeneic BDIX rats, intratumoral concentration of TGF-beta1 increases while NO concentration stays very low. Tumor regression induced by intraperitoneal injections of a lipid A is associated with a decrease in TGF-beta1 and an increase in NO intratumoral concentration. In these tumors, PROb tumor cells are the NO- and TGF-beta1-secreting cells. Using PROb cells transfected with an expression vector coding for TGF-beta1 antisense mRNA, we demonstrate in vitro that there is an inverse correlation between the amount of TGF-beta1 secreted and the ability of PROb cells to secrete NO. As the same results were obtained in the presence of an anti-TGF-beta type II receptor neutralizing antibody, and as exogenous TGF-beta1 is without any effect on NO secretion by PROb cells, TGF-beta1 apparently down-regulates NO synthesis in PROb cells by an intracellular mechanism. These results suggest that endogenous TGF-beta1 constitutes a potential target in a search for new antitumoral agents.

Topics: Activin Receptors, Type I; Animals; Carcinoma; Colonic Neoplasms; Down-Regulation; Female; Immunotherapy; Intracellular Fluid; Lipid A; Male; Neoplasm Transplantation; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Protein Serine-Threonine Kinases; Rats; Rats, Inbred Strains; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Antisense; Second Messenger Systems; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

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.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Colonic Neoplasms; Dinoprostone; Disaccharides; Drug Screening Assays, Antitumor; Indomethacin; Lipid A; Liver Neoplasms; Lung Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Peritoneal Neoplasms; Tumor Necrosis Factor-alpha

Colon carcinoma is one of the most frequent causes of cancer death in industrialized countries. The patients generally die of the metastases. In a colon cancer rat model, the authors have shown that lipopolysaccharides from Escherichia coli induced the regression of carcinomatosis and cured 20%-30% of the rats. Some synthetic derivatives of lipid A, which are less toxic than lipopolysaccharides, were injected 14 days after the tumor cells. They induced the complete regression of peritoneal carcinomatosis consisting of numerous nodules measuring 1-5 mm in 20%-30% of rats. Only compounds with three or more hydroxymyristic acid residues were effective. In vivo effects were correlated with the capacity to induce the production of interleukin 1 and tumor necrosis factor but not with the capacity to induce macrophage-mediated cytolysis. It is therefore possible to synthesize weakly toxic derivatives of lipopolysaccharides retaining their antitumoral property in vivo.

Topics: Animals; Colonic Neoplasms; Female; Interleukin-1; Lipid A; Macrophages; Male; Peritoneal Neoplasms; Rats; Tumor Necrosis Factor-alpha

Two species of neutral glycosphingolipids purified from rat colon carcinoma tissue, isoglobotetraosylceramide [GalNAc(beta 1----3)Gal(alpha 1----3)Gal(beta 1----3)Glc(beta 1----1)Cer] and a related 6-sugar "analogue" were inserted into liposomes together with lipid A (from bacterial lipopolysaccharide) and used for immunization of mice and monoclonal antibody production. The yield of hybridomas producing glycolipid-specific antibody was 5-10% using a high-dose booster schedule with liposome-inserted glycolipid. In contrast the frequency was below 0.1% (no glycolipid-binding antibodies were found) when using the previously described method of immunizing with glycolipid coated on the surface of acid-treated S. minnesota. Monoclonal antibodies were screened on the purified glycolipids used for immunization and selected for differential reactivity to the two glycolipids. A diversity of specificities was demonstrated by binding to the purified antigens, in a thin-layer chromatogram binding assay and in binding tests to tumor and normal target cells.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; Colonic Neoplasms; Glycolipids; Lipid A; Liposomes; Mice; Mice, Inbred BALB C; Rats; Rats, Inbred WF