Cure of colon cancer metastasis in rats with the new lipid A OM 174. Apoptosis of tumor cells and immunization of rats.

The antitumoral effect of the new lipid A OM 174 was investigated in a model of colon cancer in rats. Peritoneal carcinomatosis were induced in BDIX rats by intraperitoneal injection of syngeneic PROb cancer cells. The treatment started 2 weeks later, when rats had macroscopic peritoneal nodules. An antitumoral effect was first obtained with OM 174 intraperitoneally injected, then an intravenous treatment was developed. When injected 15 times intravenously, at the dose of 1 mg/kg, 2 days apart, OM 174 induced the complete regression of tumors and hemorrhagic ascitis in 90% of the tumor-bearing rats, whereas all the untreated rats died of their tumors. To our knowledge, this treatment is the most effective ever applied to macroscopic tumors. Furthermore, the treatment induced the immunization of rats since the reinjection of PROb tumor cells in OM 174-cured rats did not cause the formation of new tumors while injection of another syngenic colon tumor cells did. Only in treated rats tumors were infiltrated with lymphocytes, macrophages and fibroblasts. The treatment did not increase necrosis but generated apoptotic areas. OM 174 was not directly toxic for tumor cells, and thus the observed effect involved the host-mediated antitumor reaction. Therefore we hypothesize that OM 174 therapy induces tumor cell apoptosis, stimulates the phagocytosis of apoptotic bodies and then activates immune system by antigen presentation.

Involvement of T lymphocytes in curative effect of a new immunomodulator OM 163 on rat colon cancer metastases.

In a model of colon cancer in syngeneic rats, a new immunomodulator, OM 163, induced the complete disappearance of peritoneal carcinomatosis (nodules measuring 1-5 mm) in 41 out of 82 rats. Those results were confirmed in a survival experiment in which 3 out of 10 treated rats died free of tumour 10, 18 and 28 months after the tumour cell injection while all the untreated control rats died of their tumours within 3 months. OM 163 had a systemic effect, since injected intraperitoneally it completely inhibited the growth of lung metastases in 13 out of 20 rats. The antitumour effect of OM 163 was also observed in two rat strains on original tumours. Lymphocyte infiltration was observed in the tumours mainly constituted of CD4+ and CD8+ cells. The treatment had no effect in nude rats, confirming the involvement of T lymphocytes. Furthermore, rats cured by OM 163 were protected against a second challenge of tumour cells and in a Winn's assay, splenocytes from cured rats protected normal rats against tumour cells.

Correlation between the capacity to activate macrophages in vitro and the antitumor activity in vivo of lipopolysaccharides from different bacterial species.

The correlation between the activation of macrophages by lipopolysaccharides (LPS) from four different bacterial species and their antitumor effect in a rat model of colon cancer was investigated. The efficacy of LPS from Neisseria meningitidis (Nm), Salmonella minnesota (Sm), Escherichia coli (Ec) and Bordetella pertussis (Bp) was evaluated as the smallest concentration inducing rat peritoneal macrophages (pm psi) to produce tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6 and nitric oxide (NO). The cytokine production was measured in bioassays and NO production quantitatively with Griess reactant. Nm was the most effective LPS with concentrations of 1 ng/10(6) pm psi for the induction of TNF, IL-1 and IL-6 activities and 0.01 ng/10(6) pm psi for the induction of NO production. The range between efficacy of different LPS was broad from 1 to 10(4)-10(5) for TNF activity, 1 to 10(2)-10(3) for NO production and IL-6 activity and 1 to 10-10(2) for IL-1 activity. In vivo antitumor effect was evaluated on the growth of peritoneal carcinomatosis. Complete tumor regressions were observed, the LPS rating with respect to decreasing efficacy was Nm, Sm, Ec then Bp; Nm, Sm and Ec were very closed while Bp was not effective. These results show the correlation between the antitumor effect in vivo of LPS and their capacity to induce in vitro IL-1 activity, but not between their ability to induce NO production, TNF and IL-6 activities.

Interleukin-8 antitumour effect is associated with a local infiltration but not with a systemic activation of T lymphocytes.

In a model of colon cancer in rats (peritoneal carcinomatosis), IL-8 was found to have a highly reproducible antitumoural effect. During IL-8-induced tumour regression the infiltration of nodules by CD4+ T lymphocytes was enhanced. However, splenic lymphocytes did not proliferate in response to tumour cells in vitro. IL-8 antitumour effect was associated with a local but not with a systemic activation of T lymphocytes.

[Experimental chemotherapy of peritoneal carcinomatosis of colonic origin in rats]. / Chimiothérapie expérimentale d'une carcinomatose péritonéale d'origine colique chez le rat.

Animal models are useful in the evaluation of adjuvant or palliative treatment modalities of human colonic adenocarcinoma. In the present paper, the efficacy of 22 usual chemotherapeutic agents was evaluated in a model of peritoneal carcinomatosis of colonic origin in the BD IX rat. Mitomycin, cisplatine, carboplatine, cyclophosphamide, ifosfamide, and thiotepa were very effective agents on microscopic carcinomatosis (treatment given 3 days after an intraperitoneal inoculation of 1 x 10(6) DHD/K12/PROb cells). Intravenous administration was as effective as the intraperitoneal route, except for anthracyclines and 5-fluorouracil. Rats treated at early stages by thiotepa or cisplatin survived up to 4 months after cell injection and did not display tumor at autopsy. Administered late (15 days after cell injection), none of the drugs were able to cure the rats with carcinomatosis.

Immunotherapy of colon cancer metastases in rats with the immunostimulant OM-89.

In a model of colon cancer in rats the bacterial extract OM-89 was found to have antitumor properties. We have shown that OM-89 induced the complete regression of numerous peritoneal tumor nodules measuring 1-5 mm in 46% of rats, and inhibited the growth of microscopic tumors in 42% of rats. OM-89 was effective when injected ip at the dose of 50 mg/kg, twice a week for 2 weeks. It was ineffective at the same dose when given orally every day for 14 or 21 days. The traces of endotoxins (0.0005%) were probably not involved in the antitumor effect of OM-89. No side-effects were observed.

Antitumor effect of synthetic derivatives of lipid A in an experimental model of colon cancer in the rat.

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.

Interleukin-8 has antitumor effects in the rat which are not associated with polymorphonuclear leukocyte cytotoxicity.

The antitumor effect of lipopolysaccharides (LPS) has been observed in several experimental models and is likely to be mediated by macrophages. Stimulation of macrophages with LPS results in the release of several cytokines, including tumor necrosis factor, interleukin-1 and neutrophil-activating peptide-1/interleukin-8 (IL-8), which activates polymorphonuclear leukocytes (PMN) in vitro. Since PMN have an antitumor activity, we tested the in vivo effect of IL-8 on the growth of peritoneal carcinomatoses induced by PROb colon cancer cells in syngeneic rats. IL-8 induced a significant regression of tumors measuring 1-5 mm, and a complete regression was observed in 8 out of 40 rats in four independent experiments. IL-8 was not directly cytotoxic in vitro for tumor cells and was effective in vivo in a narrow range of doses. IL-8 had a significant chemotactic effect for peritoneal PMN in both normal and tumor-bearing rats. PMN taken from the peritoneum of tumor-bearing rats during IL-8 treatment had the same cytotoxic activity against PROb tumor cells as PMN from untreated control rats. Microscopic examinations of tumors during the treatment showed poor infiltrating by PMN. We conclude that the antitumor activity of IL-8 in this model is not mediated by PMN cytotoxicity.

Nitric oxide involvement in tumor-induced immunosuppression.

The mechanisms of immunosuppression induced by colon cancer in rats were investigated at the systemic and tumor levels. During tumor growth (after i.p. injection of rat colon adenocarcinoma cells in syngeneic BD IX rats), Con A-induced proliferation of splenic mononuclear cells decreased and nitric oxide (NO) production by splenic macrophages increased concomitantly. Incubating splenic mononuclear cells with an inhibitor of NO synthase, NG-monomethyl-L-arginine, restored lymphocyte proliferation. A low level of inducible NO synthase mRNA was detectable in tumors by Northern blotting, with a weak increase during tumor growth. The NO concentration measured in the tumor nodules increased weakly parallel to the tumor growth. Five and six weeks after tumor cell injection, tumor-infiltrating lymphocytes from disaggregated tumors did not proliferate in the presence of Con A. Addition of NG-monomethyl-L-arginine inhibited the production of NO in tumor dissociations and enhanced tumor-infiltrating lymphocyte proliferation. Glyceryl trinitrate (a NO-releasing compound) totally inhibited the lymphocyte proliferation in vitro while it slightly reduced the tumor cell proliferation. T lymphocytes were therefore more sensitive to NO than were tumor cells. Culture medium from tumor cells induced NO production by splenic macrophages, although the factor involved has not yet been identified. Furthermore, tumor cells could also play a part in NO production by tumors because the tumor cells were induced to produce NO by IFN-gamma plus IL-1. These results strongly suggest the participation of NO in the tumor-induced immunosuppression in rats.

Expression of inducible nitric oxide synthase in tumors in relation with their regression induced by lipid A in rats.

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.

Nitric oxide inhibits proliferation but increases life-span of T lymphocytes in tumour-bearing rats.

Nitric oxide (NO) has been shown to inhibit the proliferation of lymphocytes. However, in tumour-bearing rats treated with the immunomodulator OM 163, the regressing nodules were heavily infiltrated by T lymphocytes, although they contained high levels of NO. We show here that NO, while inhibiting the proliferation of lymphocytes, increased their life-span, pointing to the ambivalence of this molecule in the course of tumour growth and regression.

Evidence for control of nitric oxide synthesis by intracellular transforming growth factor-beta1 in tumor cells. Implications for tumor development.

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.