АNTIMETASTATIC EFFECT OF B. SUBTILIS IMV B-7724 LECTIN OBSERVED IN LEWIS LUNG CARCINOMA MODEL.

AIM: To study the antitumor and antimetastatic effects of B. subtilis IMV B-7724 lectin used in neoadjuvant and adjuvant settings in vivo. MATERIALS AND METHODS: Studies were performed on C57Bl/6J mice; Lewis lung carcinoma (LLC) was used as an experimental tumor. В. subtilis ІМV В-7724 lectin was administered to tumor-bearing mice or to mice which underwent surgical resection of the primary tumor. The lectin was injected subcutaneously, 10 times, at a single dose of 5 or 1 mg/kg of body weight. The standard indicators of tumor growth and metastasis were evaluated. RESULTS: Independently of the application settings, the lectin at a dose of 1 mg/kg of b.w. caused more pronounced effect than at a dose of 5 mg/kg of b.w. The administration of B. subtilis IMV B-7724 lectin to the mice with LLC in neoadjuvant setting did not cause notable antitumor effect but led to a significant decrease in the number and volume of lung metastases. The lectin administration in adjuvant setting significantly inhibited metastasis: the metastasis inhibition index reached 63.0% and 100% in the mice treated with the lectin at a dose of 5 mg/kg and 1 mg/kg respectively. The mean survival time of the treated animals significantly increased. CONCLUSION: A pronounced antimetastatic effect of B. subtilis IMV B-7724 lectin administered in an adjuvant setting was demonstrated.

Influence of bacterial lectin on key regulatory links of functional activity of macrophages in mice with Ehrlich carcinoma.

BACKGROUND: Recent studies have shown the potential of using different approaches for immunotherapy in cancer treatment. Macrophages (Mph) are one of the promising targets for immunotherapy. AIM: To investigate changes in the functional activity of Mph in mice with Ehrlich carcinoma by nitric oxide (NO)/arginase (Arg), IRF4/IRF5 and STAT1/STAT6 ratios caused by administration of lectin from B. subtilis IMV-7724. MATERIALS AND METHODS: From the 2nd day after Ehrlich carcinoma inoculation into female Balb/c mice, lectin from B. subtilis IMV B-7724 (0.02 mg/mouse) was administered for 10 days. The peritoneal Mph were isolated on days 14, 21, and 28 after tumor transplantation and their functional state (NO production, Arg activity and cytotoxic activity) was examined. The levels of mRNA expression of transcription factors STAT-1, STAT-6, IRF5, IRF4 were evaluated. RESULTS: In lectin-treated animals with Ehrlich carcinoma, the functional state of Mph (NO/Arg ratio, index of cytotoxic activity) was maintained at the level of intact mice exceeding the values in untreated animals with Ehrlich carcinoma at late terms of tumor growth (21, 28 days). Analysis of mRNA expression levels of transcription factors in these animals showed a significant increase (p < 0.05) in the ratio of STAT1/STAT6 on the day 21 and IRF5/IRF4 on day 28 of tumor growth compared to that in untreated mice. CONCLUSIONS: Administration of lectin from B. subtilis IMV B-7724 to mice with Ehrlich carcinoma led to the prevalence of Mph exhibiting the functional properties of M1 type at late-term tumor growth. The transcription factors of the STAT and IRF signaling pathways are involved in the process of Mph polarization induced by lectin from B. subtilis IMV B-7724.

Oxamate, an inhibitor of lactate dehydrogenase, can stimulate M2 polarization of peritoneal macrophages in mice with Lewis lung carcinoma.

BACKGROUND: Inhibition of aerobic glycolysis of cancer cells is considered a promising therapeutic strategy for the treatment of neoplasms. Some inhibitors of energy metabolism can affect not only tumor cells but also the functional polarization of tumor-associated macrophages, which may either enhance the antitumor effect of such agents or impair their antitumor efficacy. AIM: To investigate the effect of oxamate, a lactate dehydrogenase (LDH) inhibitor, on the polarization of peritoneal macrophages (PMP) in both intact mice and mice with transplanted Lewis lung carcinoma (LLC). MATERIALS AND METHODS: The low-metastatic LLC variant, LLC/R9, was transplanted to female C57Bl/6 mice. Sodium oxamate was used as the test agent at concentrations of 0.02, 0.2, and 2 mg/ml. Macrophage polarization in tumor-bearing mice was estimated on day 23 after tumor transplantation by assessing nitric oxide (NO) production and arginase activity as functional indices of PMPs polarization. RESULTS: Oxamate can affect the functional polarization of PMPs in both intact mice and animals with transplanted LLC/R9. Oxamate in all studied concentrations changed the markers of PMPs polarization in intact mice (decreasing NO levels and activating arginase activity) that indicated the stimulation of M2 polarization. In tumor-bearing animals, stimulation of M2 polarization is observed at low concentrations of oxamate (0.02 mg/ml), but its high concentrations (2.0 mg/ml) causes M1 polarization, which is characterized by three-fold increase in the level of NO and a decrease in the level of arginase activity. CONCLUSION: Oxamate, an inhibitor of LDH, can stimulate M2 polarization of peritoneal macrophages of mice bearing LLC in a dose-dependent manner.

Functions of tumor-associated macrophages and macrophages residing in remote anatomical niches in Ehrlich carcinoma bearing mice.

BACKGROUND: The impact of growing tumor on polarization and functions of tumor-associated macrophages is well known while its influence on residential macrophages occupying different anatomical niches reminds to be elucidated. AIM: To study changes in polarization and functions of macrophages isolated from discrete anatomical niches in tumor-bearing mice at different stages of tumor growth. MATERIALS AND METHODS: Ehrlich carcinoma was transplanted intramuscularly to Balb/c male mice. On days 7, 14, 21 and 28 after tumor transplantation, macrophages from tumor tissue, peritoneal cavity and spleen were isolated and analyzed. Nitric oxide production was measured by standard Griess reaction, arginase activity was determined by the measurement of urea, reactive oxygen species production was checked using NBT dye reduction assay and electron paramagnetic resonance spectroscopy, cytotoxic activity was estimated in MTT-assay. RESULTS: Independently of their localization in different anatomic niches, macrophages in mice with transplanted Ehrlich carcinoma gradually lose their tumoricidal activities while arginase activity is upregulated. This indicates the shift of polarization from M1-like towards M2-like phenotype. CONCLUSION: Our findings demonstrated that growing tumor could be able to subvert functioning of macrophages at the systemic level.

Macrophages - a perspective target for antineoplastic immunotherapy.

The review discusses the data on the functional/phenotypic M1/M2 types of macrophages and their chimeric forms, the molecular mechanisms of polarization of these cells, and their role in the development of malignant tumors. Information on the prognostic value of the presence (density and location) of M1/M2 cells in tumor tissue is analyzed. Our own results evidence on the necessity of determination of the functional/phenotypic state of M1/M2 macrophages from different biological niches in the dynamics of tumor growth, in particular in terms of NO level and arginase activity.

The effects of early postoperative immunization with xenogeneic embryo proteins on Lewis lung carcinoma model.

AIM: To investigate the effect of chicken embryo proteins (CEP) as a prototype of xenogeneic vaccine on immune reactions in mice immunized after Lewis lung carcinoma (LLC) surgical removal. MATERIALS AND METHODS: C57Bl male mice were immunized on days 1, 8, and 15 after surgical removal of LLC. The immune response was assessed on days 7, 14, 21 and 28 after tumor resection. Cytotoxic activity of natural killer cells (NK) and cytotoxic T-lymphocytes as well as antibody dependent cellular cytotoxicity was estimated in MTT-assay; specific antibodies were detected in ELISA; lymphocyte proliferation was tested in reaction of in vitro blast transformation. RESULTS: None of the immunized mice developed LLC metastases. Immunization with CEP seems to prevent the potential decrease in NK cell cytotoxic activity and spontaneous blast transformation activity of lymphocytes following the surgically induced stress. Further research on improving immunization schedule and elucidating the mechanisms of NK modulation with CEP is needed.

Anticancer effect and immunologic response to xenogeneic embryonic proteins in mice bearing Ehrlich solid carcinoma.

AIM: To investigate anticancer and immunologic effects of chicken embryonic proteins (CEP) in mice bearing Ehrlich solid carcinoma. MATERIALS AND METHODS: The study was carried out on male Balb/c mice bearing Ehrlich solid carcinoma. The immunizations were performed after the tumor transplantation. The immune status was assessed on days 7, 14, 21 and 28 after the tumor challenge. Cytotoxic activity (CAT) of macrophages (Mph), natural killer cells (NK), cytotoxic T-lymphocytes (CTL) and blood serum, as well as the influence of the blood serum on immune cells activity was checked in MTT-assay; Mph's cytochemical activity was tested in NBT-assay; Ehrlich antigen-specific or CEP-specific antibodies were detected in ELISA-assay; medium size circulating immune complexes (CIC) were detected in reaction of 4.5% polyethylene glycol precipitation. RESULTS: The immunization resulted in tumor growth suppression and significant 25.64% prolongation of the survival time. In both control and immunized mice with transplanted tumors antibodies specific to Ehrlich carcinoma antigens and to CEP were detected, but antibody response was more balanced in the treatment group. In the treatment group both cytochemical and CAT of Mph was moderately activated and well preserved until late stages of tumor development; CAT of NK and CTL remained in the range of the intact mice until day 28 after the tumor transplantation. The immunized mice were well protected from accumulation of CIC and suppressive activity of autologous blood serum. CONCLUSION: Collectively, our data indicate that CEP can elicit immunomodulating and immunoprotecting effects sufficient to provide tumor growth inhibition. The further elaboration of a xenogeneic anticancer vaccine based on CEP is warranted.

Effect of enterosorption on paraneoplastic syndrome manifestations in mice with highly angiogenic variant of Lewis lung carcinoma.

AIM: To study the correcting effects of microgranulated HSGD enterosorbent on hematological, morphological and biochemical indices of paraneoplastic syndrome in mice with highly angiogenic variant of Lewis lung carcinoma LLC/R9. METHODS: The study was performed on male С57/ВL6 mice with transplanted LLC/R9. Enterosorbent HSGD was administered daily at a dose of 0.625 g/kg for 2 weeks starting from 7(th) day after tumor cell transplantation. When enterosorption was completed, an analysis of peripheral blood, biochemical indices and morphological structure of tumor, lung, liver, spleen and thymus was carried out by standard methods. RESULTS: It has been shown that administration of enterosorbent did not affect LLC/R9 growth but resulted in nearly two fold decrease of the volume of lung metastases (p < 0.05). Erythrocyte number and hemoglobin level were higher by 30.0% (p < 0.05) and 23.3% (p < 0.05), respectively, in mice treated with enterosorbents as compared to untreated animals. In addition sorbent treatment completely normalized the thrombocyte index resulting in elevation of platelet number by 54.5% (p < 0.01) up to their level in intact mice. The morphological examination of liver and biochemical analysis of peripheral blood evidenced on significant positive correcting effect of enterosorption on histological structure of this organ and its functional activity. Normalization of total proteins and serum albumin level as well as significant decrease of total lipid concentration by 29% (p < 0.01) in blood of treated mice were observed. CONCLUSION: Positive influence of microgranulated carbon sorbent on some hematological, morphological and biochemical indices of tumor associated symptoms in LLC/R9-bearing mice denotes that enterosorption-based therapy can be considered as a prospective treatment for correction of some paraneoplastic syndrome signs in cancer patients.

Anticancer effectiveness of vaccination based on xenogeneic embryo proteins applied in different schedules.

THE AIM: To evaluate anticancer activity of vaccination with chicken embryo proteins (CEP) applied in different schedules. MATERIALS AND METHODS: C57Bl mice were vaccinated with CEP before (prophylactic schedule) or after (different therapeutic schedules with or without preliminary tumor removal) the Lewis lung carcinoma cells transplantation. The latent period of tumor development, tumor volume and metastasis rate were evaluated. RESULTS: Potent antimetastatic effect of CEP-based vaccination was seen in case of therapeutic regimen after primary tumor removal. The metastasis inhibition index (MII) reached 96.9 and 97.8% on 18(th) and 34(th) day after tumor removal, respectively. When CEP vaccination was performed in the settings of therapeutic regimen without primary tumor removal the anticancer effect was evident only if vaccinations started as early as 24 h after the cancer cells injections. The highest MII achieved in such condition was 77.6%, tumor volume in the group of vaccinated animals was by 53.1-42.1% lower than in the control tumor-bearing mice. CEP vaccination before tumor challenge (prophylactic immunization) led to a statistically significant prolongation of the latent period of tumor development, a reduction of tumor volume (35.8-48.8% compared to control unvaccinated mice) and a marked inhibition of metastasis (MII was 71.1%). CONCLUSION: Vaccination based on CEP exhibited both prophylactic and therapeutic anticancer effects. The last one is more pronounced when the vaccination starts shortly after the primary tumor resection.

Cytotoxic activity of immune cells following administration of xenogeneic cancer vaccine in mice with melanoma B-16.

AIM: To study the effects of xenogeneic cancer vaccine (XCV) developed on the basis of nervous tissue antigen from rat embryo of late gestation period and protein-containing metabolite of Bacillus subtilis with molecular weight of 70 kDa, on specific and unspecific antitumor reactions of cellular and humoral chains of immune system, and to analyze possible mechanisms of its antimetastatic action. MATERIALS AND METHODS: XCV was administered triply with 3-day intervals after surgical removal of experimental melanoma В-16 in C57Bl/6 mice. Cytotoxic activity (CTA) of splenocytes against target cells К-562 as well as CTA of splenocytes, peritoneal macrophages (PM) and blood serum against melanoma В-16 target cells were determined using МТТ test. The content of circulating immune complexes (CIC) in blood serum was evaluated by precipitation reaction. RESULTS: Immunologic effects of XCV vaccination in experimental animals with surgically removed melanoma B-16 in comparison with similarly treated unvaccinated mice were as follows: prevention of medium molecular weight CIC accumulation in blood serum during all observation period, significant increase (р < 0.05) of CTA of effectors of unspecific antitumor immunity (natural killer cells - NK - by 25.5 ± 1.7 vs 12.5 ± 5.4%, and PM - by 37.3 ± 0.6 vs 32.0 ± 0.9%, respectively) at 37(th) day after the surgery, and also preservation of functional activity of specific cytotoxic lymphocytes at the level of intact control. CONCLUSION: The results of the study allow propose that antimetastatic effect of XCV vaccination could be based on increased CTA of NK and PM, and preservation of CTL functional activity at late terms after surgical removal of B-16 primary tumors.