Cytostatic effect of cyclophosphane on esophageal tumors and epithelium of mice

Investigation of cytostatic activity of cyclophosphamide in sarcoma 37 and esophagus epithelium in albino mice with respect to the diurnal rhythm of mitotic activity and the number of labeled nuclei was performed. Apparently the tumour cells in the G1-phase and at the beginning of the S-phase of the mitotic cycle were the most sensitive to the inhibitory effect of this drug. During the completion of the DNA-synthesis period the cell resistance to the action of the cytostatic increased. Cells at the G1-phase of the mitotic cycle were sensitive to the inhibitory action of cyclophosphane in the esophageal epithelium.     

Myelopeptides: Bone marrow regulatory mediators

Bone marrow cells of various animal species and men produce a group of bioregulatory peptides called myelopeptides (MPs). A highly purified MP fraction and some individual molecules have been isolated from the supernatant of porcine bone marrow cell cultures by reverse phase chromatography.MPs have a wide spectrum of functional activities: immunoregulatory, differentiating and opiate-like. They evoke 2–5-fold stimulation of antibody production to various antigens. They correct some immune defects in MRL/lpr mice with spontaneous autoimmune disorders that results in 2-fold prolongation of the life span of these mice. MPs influence the differentiation of bone marrow and peripheral blood cells derived from healthy and leukemic donors. They induce terminal differentiation in the leukemic human HL-60 cell line. MPs also show an effect on pain sensitivity.A new immunocorrective drug Myelopid has been developed on the basis of MP mixtures. This drug is effectively used in Russia both in medicine and veterinary practice for prophylaxis and treatment of diseases accompanied by immunodeficiency.Two individual MPs were isolated and identified: Phe-Leu-Gly-Phe-Pro-Thr (MP-1) and Leu-Val-Val-Tyr-Pro-Trp (MP-2). MP-1 displays immunoregulatory activity; MP-2 abolishes the inhibitory effect of leukemic cells on T-lymphocyte functional activity.MPs seem to provide not only immunoregulation but also to participate in complex interactions between different systems in the organism.     

Peculiarities of immunocorrective effects of the bone marrow regulatory peptides (myelopeptides)

Myelopeptides (MPs) are low-molecular-weight immunoregulatory peptides of bone marrow origin. The peculiarities of their immunoregulatory effects are demonstrated with two of the six synthesized MPs, MP-1 (Phe-Leu-Gly-Phe-Pro-Thr) and MP-2 (Leu-Val-Val-Tyr-Pro-Trp). It is shown that MP action is directed to the damaged links of immunity. MP-1 enhances a decreased level of antibody production in cyclophosphamide (Cy)-treated mice, but does not influence the antibody formation in normal animals. MP-2 inhibits the tumor growth more in a tumor-bearing organism as the tumor size gets larger, insofar as MP-2 antitumor effect is concerned, by its ability to recover functional activity of T lymphocytes suppressed by tumor products. Selective immunocorrective effects of MPs are based on ligand-receptor interactions. Using FITC-labeled MP-1 and [3H]-labeled MP-2, specific binding of these peptides with appropriate cell populations is shown. The cytofluorimetric analysis revealed a target cell for MP-1--CD4+ T lymphocyte (T helper). The data obtained suggest that MPs are endogenic immunoregulators which participate in the maintenance of immune homeostasis.     

Myelotoxicity of epirubicin]

Epirubicin (pharmorubicin, India), an antitumor antibiotic of the anthracycline group, was studied in regard to its effect on peripheral blood, bone marrow and lymphoid organs (the thymus and spleen) of CBA mice after its intraperitoneal administration in a single dose equal to the MIC (7.8 mg/kg) and in a course dose (1/5 of the MIC 5 times a day). The cytogenetic impairments induced by the cytostatic were estimated on metaphase plates with the bone marrow specimens and by counting the peripheral blood erythrocytes with micronuclei (the micronuclear test). It was shown that epirubicin induced cytogenetic disturbances in the hemopoietic cells within the first 72 hours. The antibiotic had a marked reversible effect on the erythroid population and lymphoid tissues and a moderate toxic action on the granulocyte population. The antibiotic did not affect thrombocytopoiesis. The single administrations had a more pronounced and prolonged myelotoxic and lymphotropic effect.     

Endogenous immunoregulatory peptides (myelopeptides): structure, function, and mechanism of action

Previously unknown bioregulators from bone marrow, myelopeptides, were isolated, identified, and synthesized, and their biological properties and mechanism of action were studied in detail. Phe-Leu-Gly-Phe-Pro-Thr (MP-1) manifests an immunocorrecting effect by restoring the level of antibody production in animals suffering from immunodeficiencies of various etiologies; Leu-Val-Val-Tyr-Pro-Trp (MP-2) manifests an antitumor effect by abolishing the inhibitory action of tumor cells on the functional activity of T-lymphocytes; Leu-Val-Cys-Tyr-Pro-Gln (MP-3) stimulates phagocytosis by macrophages and, in this way, protects animals from infections caused by pathogenic microorganisms; and Phe-Pro-Arg-Ile-Met-Thr-Pro (MP-4) is a new factor of cell differentiation inducing terminal cell differentiation in the HL-60 and K-562 leukemic cell lines.     

Synthesis and antitumor properties of the myelopeptide MP-1

The bone marrow-derived peptide Phe-Leu-Gly-Phe-Pro-Thr (MP-1) has been synthesized by the classical methods of peptide chemistry in solution, and its antitumor properties have been studied. It has been shown that MP-1 enhances the efficacy of the cytostatic therapy of lympholeukosis P388, increases the latent period of the growth of P388 tumors implanted in irradiated mice, and reduces the recurrence of the breast adenocarcinoma Ca-755 in mice after the surgery.     

Effect of autologous blood irradiated in isolation on the restoration of leukopoiesis in an experimental model of prolonged leukopenia

Reinfusion of irradiated (220 Gy) isolated blood (IIB) was shown to accelerate leukopoiesis restoration in conditions of myelodepression induced by the injection of cyclophosphane. Restoration of leucocyte count in the peripheral blood was preceded by the increase in DNA synthesis and bone marrow cellularity. Reinfusion of IIB also promoted a more rapid restoration of cellularity of lymphoid organs with cAMP predominating therein. The comparison of the processes under study in time permits to assume that the stimulatory effect of IIB is related to activation of proliferation and stimulation of cell maturing in bone marrow and lymphoid organs.     

Effect of doxorubicin on the functional state of the mononuclear phagocyte system]

The response of the system of mononuclear phagocytes (SMP) to doxorubicin, an antitumor antibiotic, most widely used in oncological care, was studied. It was shown that a single intraperitoneal administration of doxorubicin to CBA mice in the maximum tolerance doses induced suppression of absorptive SMP capacity and increased IL-I secretion by the bone marrow and peritoneal macrophages both in the stimulated and spontaneous tests in early periods after cytostatic administration. There was a significant rise in the ability of SMP bone marrow elements to respond to the macrophage activating factor, as well as an increase in the cytotoxic activity of bone marrow and peritoneal macrophages.     

No effects of levamisole on cytotoxic drug-induced changes of human granulopoiesis.

The effect of Levamisole on the human granulopoiesis was studied in patients randomized to receive, in addition to adjuvant chemotherapy for primary breast cancer, either no other treatment or additional unspecific immune therapy with Levamisole. The reaction of granulopoiesis to the cytostatic drugs, as characterized by changes of peripheral blood polymorphonuclear neutrophils (PMN), functional bone marrow granulocyte reserve, serial bone marrow cytology, and granulopoietic stem cells (CFU-C) in marrow and blood, was not affected by administration of Levamisole. The data support the concept that Levamisole has no direct effect on human bone marrow granulopoiesis, but that an allergic mechanism is involved in the pathogenesis of Levamisole-induced agranulocytosis. The expectation that Levamisole exerts a beneficial effect by stimulation of the granulopoiesis, as previously suggested for BCG and Corynebacterium parvum, could not be substantiated in our studies.     

The effect of different infectious agents on the intensification of hematopoiesis during immunosuppression

The action of Staphylococcus aureus, Escherichia coli and Herpes simplex virus on the hemopoiesis of mice with cytostatic myelosuppression was studied. The study revealed that the infection of the animals simultaneously with the action of cyclophosphamide considerably activated the processes leading to the restoration of hemopoiesis due to an increase in the mitotic activity of hemopoietic cells, the accelerated differentiation of hemopoietic precursor cells which could survive the cytostatic action and an increase in the functional activity of the hemopoiesis-producing microenvironment.     

Antimutagenic effect of Agaricus blazei Murrill mushroom on the genotoxicity induced by cyclophosphamide

Agaricus blazei Murrill extracts have previously been shown to have anticarcinogenic and antimutagenic properties. These results suggest that antimutagenic activity, besides the modulation of the immune system, might be involved in the anticarcinogenic action of A. blazei. To investigate the possible antimutagenic effect of A. blazei in vivo, we evaluated its effect on clastogenicity induced by cyclophosphamide (CP) in mice, using the micronucleus test in bone marrow (MNPCE) and in peripheral blood (MNRET). Male Swiss mice were treated with CP (25 or 50mg/kg i.p.) or with CP plus mushroom solution at three different temperatures: 4, 21, and 60 degrees C. Aqueous solution of a mixture from various lineages of the mushroom inhibited induction of micronuclei by CP in bone marrow and in peripheral blood of mice. In contrast to the mixture of lineages, a single isolated lineage did not lead to a reduction of CP-induced MN frequencies in either bone marrow or blood cells of mice. The results suggest that under certain circumstances these mushrooms exhibit antimutagenic activities that might contribute to an anticarcinogenic effect.     

Myelopeptide MP-5 and fluorescent derivatives: Synthesis and biological activity

The Val-Val-Tyr-Pro-Asp bone marrow peptide (MP-5) and its analogue (MP-5-Lys) were synthesized. Fluorescent derivatives, Ftc-MP-5 and MP-5-Lys(Ftc), were prepared. The iological activity of MP-5 and MP-5-Lys was studied in vitro and in vivo. The MP-5 peptide caused 60–84% inhibition of growth of the following mouse cancers: lymphatic leukemia P388, melanoma B-16, and cervical carcinoma CUC-5. These peptides also restored functional activity of T-lymphocytes that was inhibited by metabolic products of the HL-60 leukemic cell line. MP-5-Lys(Ftc) was shown to preserve the functional properties of MP-5 towards T-lymphocytes, but Ftc-MP-5 was practically inactive.     

Action of carminomycin on leukemic cell proliferation and some problems of its use in the combined therapy of leukemias

The effect of carminomycin on the mitotic cycle of the cells of the transplantable leukemia L-1210 and the therapeutic activity of other antitumor drugs, such as phopurine and cyclophosphane was studied on mice BDF1. It was found that the cells in phases S and G2 of the mitotic cycle were most sensitive to carminomycin. Transfer G1--S and phase G1 were characterized by resistance to the antibiotic effect. When carminomycin was used in combination with phopurine or cyclophosphane, clear dependence of the therapeutic efficacy on the treatment scheme was noted. Simultaneous administration of all the three drugs resulted in potentiation of the antileukemic effect. An analogous effect was observed when carminomycin was administered prior to phopurine or cyclophosphane. When the order of the drugs use was reverse, the efficacy of the combined therapy was significantly lower than the summation antileukemic effect of the drugs.     

Regulation of leucogenesis by extracellular ubiquitin in rodents after chemically induced inhibition

To study the influence of intraperitoneal injected extracellular ubiquitin on regeneration of leucopoiesis calculation of nuclear cell count in bone marrow (BM) and peripheral blood (PB) smears stained with azure-eosin was performed. In the first, control group of animals inhibition of haematopoiesis achieved by means of 100 mg/kg cyclophosphamide LD₅₀ 50–200 mg/kg injection. Bone marrow and peripheral blood samples from the first group of rats had been taken at 24, 48, 72, 96 and 168 h points after injection of cytostatic. Animals of the second, test group were injected by 200 μg/mL ubiquitin 72 h later after cytostatic injection. Our experiments revealed that ubiquitin makes corrections in regeneration of leucopoiesis and leads to normalisation of the process. Ubiquitin regulates stem cell activity, normalizes the release of functional cells into bloodstream, supposedly retains progenitor cells in zones of differentiation and maturation, and restores the nuclear cell ratio in PB and BM. We suppose that obtained results are important for elucidation of new pathways of ubiquitinylation and give us possibilities to find new therapeutics for regeneration of leucopoiesis that is very essential for treatment of radiated bone marrow and chemotherapeutic side effects in cancer patients.     

Vascular endothelium: target or victim of cytostatic therapy?

Chemotherapy continues to be the main therapeutic approach in the treatment of hematological malignancies including acute leukemia. Generally, chemotherapy is used to eliminate cancer cells and to restore normal bone marrow function. Simultaneous action of cytostatic drugs on bone marrow angiogenesis decreases the formation of new capillaries and improves therapeutic effect. However, chemotherapeutic agents may also be cytodestructive for cellular elements of other tissues, particularly the vascular endothelium, which can lead to various cardiovascular complications. In this work, we studied the effects of 2 cytostatic drugs, cytosine arabinoside (ara-C) and daunorubicin (DNR), on cultured human vascular (i.e., umbilical) endothelial cells (ECs). Ara-C and DNR were added to cultured cells at concentrations ranging from 1 ng/mL to 100 microg/mL. Drug effects were studied using phase-contrast microscopy, cell viability tests, BRDU incorporation, immunohistochemistry, flow cytometry, and cell cloning. At various concentrations, ara-C and DNR are able to induce morphological and functional changes in cultured cells related to either cytostatic or cytotoxic action. Moreover, ara-C-treated cultured cells displayed significant disturbances in cell adhesion molecule expression and interaction with blood leukocytes. Preliminary data obtained on acute leukemia patients undergoing standard cytostatic therapy ("7+3" regimen) have shown that concentration of the circulating ECs was significantly increased compared with the control group and could be as high as 500-1500 cells/mL of blood. Results obtained suggest that anticancer chemotherapy may induce systemic damage of vascular endothelium related to massive cell loss and (or) alterations of endothelial function.     

Role of H2-calponin in regulating macrophage motility and phagocytosis

The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo(R) cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo(R) cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.     

Late effects of the hematotoxic action of antineoplastic antibiotics of the anthracycline group

The morphological and functional states of hemopoiesis at late periods (up to 6 months) after using daunorubicin, carminomycin and doxorubicin, antitumor antibiotics of the anthracycline group in doses of 1/10 LD50 for 10 days were studied on 764 noninbred rats and 240 BALB/c mice. On various compensatory-functional models of hemopoiesis strain there was shown persisting inhibition of hematopoietic tissue functional activity and limited reserve reactions of granulocyto- and erythropoiesis 3 and to a lesser extent 6 months after the cytostatic action.     

Regulation of the peritoneal macrophage functional activity by the MP-5 and MP-6 myelopeptides under stress

In mice, two-hour immobilization stress inhibited zymosan-induced production by macrophages of the oxygen radicals and cytokine IL-1β. After myelopeptides MP-5 and MP-6 were administered into mice, the stress-induced inhibition of the reactive oxygen species (ROS) and IL-1β was abrogated. MP-5 peptide stimulated spontaneous ROS production by macrophages and reduced IL-10 production under stress. Thus, under in vivo conditions and under stress, the effect of MP-5 and MP-6 myelopeptides modulates the peritoneal macrophage activity.     

Inhibition of normal granulopoiesis by cytostatic agents.

Rabbits were treated with cyclophosphamide and 5-fluorouracil, myelosuppressive cytostatic agents, applied with a single dose of 1/3 LD50 or daily doses of 1/30 LD50 given for 14 days. Functional tests for evaluation of granulopoiesis were regularly performed at standard intervals and were following: leukocytosis, bone marrow picture with mitotic index, 3H-thymidine incorporation in vitro followed by autoradiography of labeled promyelocytes and myelocytes, serum lysozyme activity, mobilization of granulocyte reserve pool by staphylococcal alpha-toxin, cytochemistry of granulocytes, phagocytosis ability and Nitro-BT reduction. It has been found that 6-10 days after application of cytostatics, a marked depression of proliferation of young granulocyte forms and lowered reserve pool, are regularly observed. This was followed by spontaneous regeneration of granulopoiesis. No changes were noted in functional tests of mature granulocytes in peripheral blood. It is suggested that for investigation of the impairment of granulopoiesis after application of cytostatic agents, most suitable is evaluation of mobilization of the bone marrow reserve pool, lysozyme activity in blood serum and labelling of promyelocytes and myelocytes with 3H-thymidine in vitro.     

Role of hematopoietic cells-precursors in the regeneration of hematopoiesis after cytostatic action]

Cyclophosphamide was injected at a dose of 150 mg/kg once to CBA mice and caused expressed hypoplasia of bone marrow hemopoiesis (decreased myelokaryocyte count, CFU-S and CFU-D) from day 1 of the experiment. The rise of CFU-S (8) and CFU-S (12) to a baseline level was noted on day 4 (the time of intensive regeneration processes), whereas CFU-D levels did not change. It is suggested that cytostatic action may trigger "shunt" hemopoiesis mechanisms which enhance differentiation of primitive hemopoietic cells and proliferative activity of mature hemopoietic elements.