Antineoplastic drug NSC631570 modulates functions of hypoxic macrophages.

Hypoxia is an important factor in the macrophages microenvironment. Many physiological and pathological processes including solid tumor development are characterized by both low oxygen content and presence of macrophages. Tumor-associated hypoxia causes alternative polarization of macrophages in tumor tissue and transformation of these cells into the allies of a malignant neoplasm. The aim of the work was to investigate the effect of NSC631570, a cancer-selective drug that is known to selectively accumulate in the tumor tissue, on hypoxic macrophage function. Murine peritoneal macrophages (PMs) were subjected to hypoxia (3% O2). Nitrite level was assayed by the Griess reaction. Arginase activity was measured by colorimetric method. ROS generation and phagocytosis was estimated by flow cytometry. O2(-) generation was assayed by the NBT reduction method. HMGB1 expression was determined by ELISA. 42 h hypoxia caused alternative polarization of murine PMs with significant arginase prevalence. NSC631570 repolarized arginine metabolism of hypoxic macrophages to NOS dominant and activated their pro-inflammatory functions: recovered ROS production and increased alarmin release NSC631570 can restore pro-inflammatory functions of macrophages, alternatively polarized by hypoxia.

[Cysteine proteinases and their inhibitors in the development of mouse HA-1 hepatoma and antineoplastic therapy]. / Tsisteinovye proteinazy i ikh ingibitory pri razvitii myshinoi HA-1 gepatomy i protivoopukholevoi terapii.

Development of murine HA-1 hepatoma was accompanied by increased activity of cathepsin B (in ascitic cells), cathepsin D (in ascitic fluid) and increased activity of procathepsin B. There were some changes of cysteine proteinases in liver and spleen, not involved directly into tumor growth. The most prominent changes included the decreased level of cysteine proteinase inhibitors cystatin C and stefin A in ascitic cells (and to a lesser degree in liver tissue). During tumor development serum cystatin C concentration decreased by 3-times compared to intact mice. Treatment by antitumor drug Ukraine increased life span of mice with HA-1 hepatoma (transplanted intravenously), decreased the increment of tumor weight. In ascite such treatment caused a decrease of number of tumor cells and an increase of number of macrophages. Ukraie (administered once or 5-times in a dose of 0.5 mg per mice) increased cystatin C level, revealing protective mechanism of action.

Clinical aspects of cancer treatment and new biochemical mechanisms of the drug Ukrain.

A random group of 50 patients in tumor stages T1-3N0-2M0 was selected from breast cancer patients and given Ukrain therapy by intravenous injection. Twenty-five patients received a total dose of 50 mg Ukrain (5 mg every second day, 10 injections altogether). Twenty-five patients received a total dose of 100 mg Ukrain (10 mg every second day, 10 injections altogether).

Preliminary results of individual therapy of chronic hepatitis C by Ukrain and interferon-alpha.

The effects of Ukrain and recombinant human interferon-alpha 2b (IFN) on the state of the thiol-disulfide ratio (SH/SS) of the blood (Russian Federation patent no. 2150700) were studied in vitro using the amperometric titration method. The blood of 73 chronic hepatitis C (CHC) hepatitis C virus (HCV)-RNA-positive patients was examined. Ukrain was tested in doses of 0.05-2.0 micrograms/ml and IFN in 20-1000 U/ml of blood. After in vitro examination, 59 patients were treated: 28 with Ukrain and 31 with IFN. The first group of 16 patients (including eight with HCV genotype 1b) was treated with individually selected optimal doses of Ukrain (0.5-2.5 mg every second day). The second group of 12 patients was treated with doses of 2.5 mg Ukrain independent of in vitro test results. The third group of 31 patients was treated with individually selected optimal doses of IFN (0.5-2 MU 3 times a week). It was found that 79.4% of CHC patients were sensitive to Ukrain in vitro and 65.1% were sensitive to IFN. CHC patients with genotype 1b were sensitive to IFN only in 16.7% of cases while the figure for Ukrain was 92.3%. CHC patients with other HCV genotypes (3, 1a, 2) were sensitive to Ukrain in 86.7% of cases and to IFN in 70.6%. After 1 month of individual therapy with Ukrain, 87.5% of CHC patients, including six of eight cases with HCV genotype 1b, became PCR-HCV negative. In the group receiving the standard dose of Ukrain, virological response was only 33.3%. After 1 month, 74.2% of CHC patients treated with individual doses of IFN became PCR-HCV negative and after 3 months 90.3% were PCR-HCV negative. The prognostic significance of the method for screening preparations for the treatment of CHC patients was 89.8%. Treatment with Ukrain was without serious negative effects and the number of side effects of IFN in individual therapy was significantly reduced. Ukrain can be used in the treatment of CHC patients, alone or in combination with IFN preparations; in the cases with HCV genotype 1b Ukrain seems more promising than IFN. Individual therapy with Ukrain and IFN increased the efficacy of treatment 2.5-fold in comparison with standard monotherapy with the same preparations, significantly decreased the number of side effects and dramatically improved cost-effectiveness.

Cysteine proteinase inhibitor level in tumor and normal tissues in control and cured mice.

Cystatin C is the best known extracellular endogenous cysteine proteinase inhibitor and has been studied as a possible index of tumor growth and as a marker of the effectiveness of antitumor therapy. The aim of this study was to evaluate cystatin C concentrations in murine tumor tissues (compared with other organs not directly involved with tumor development, such as the liver and spleen) during treatment with several antitumor drugs (Ukrain and/or cyclophosphane). Cystatin C concentrations in murine tissues and biological fluids was determined by enzyme-linked immunosorbent (ELISA) assay. The cystatin C ELISA test is a sandwich immunoassay, which uses immobilized rabbit antihuman cystatin C Pab and mouse antihuman cystatin C Mab-HRP (monoclonal antibodies, conjugated with horseradish peroxidase). We observed decreased serum cystatin C concentrations compared with controls in all nontreated tumor models: HA-1 hepatoma (solid and ascitic forms), lung adenocarcinoma (solid and ascitic forms) and LS lymphosarcoma. In the ascitic fluid of mice with HA-1 hepatoma the cystatin C concentration was much lower than in the serum of the same mice (about 20-fold lower). In the HA-1 model of hepatoma cells cystatin C concentration decreased about 2-3-fold compared with the control (intact liver) and Ukrain significantly increased the cystatin C concentration. Cyclophosphane treatment of LS lymphosarcoma significantly increased the cystatin C concentration in serum. Cyclophosphane treatment (50 mg/kg, single injection) increased cystatin C by up to 8-fold more in tumor issue. Ukrain treatment of LS lymphosarcoma was also followed by increased levels of cystatin C in tumor tissue (4-fold); cyclophosphane plus Ukrain had a similar positive effect. In the group with LS lymphosarcoma Ukrain or cyclophosphane plus Ukrain treatment induced a significant increase in cystatin C concentration in liver. Liver cystatin C concentration decreased in the HA-1 hepatoma group and treatment with Ukrain or carboxymethylated beta-1, 3-glucan (CMG) increased this index in both groups. Spleen cystatin C concentrations decreased about 5-fold in LS lymphosarcoma compared with controls and combined treatment with cyclophosphane plus Ukrain restored the index to the normal value. We can conclude that both murine tumors studied were characterized by low cystatin C concentrations in tumor tissues and decreased cystatin C concentrations (to a lesser degree) were also observed in liver and spleen as a result of the "toxic" effect of tumor bearing. Effective treatment in all cases (especially with Ukrain or a combination of cyclophosphane plus Ukrain) induced a significant increase in cystatin C. Obviously, the decrease in cystatin C concentration predominantly in tumor tissue was connected with tumor development and restoration of cystatin C level may be used as a marker of efficacy of antitumor therapy.

Influence of Ukrain on the nuclear thyroid hormone receptors after short-term gamma-irradiation.

The ability of Ukrain, a semi-synthetic alkaloid thiophosphoric acid derivative (NSC-631579), to influence thyroid hormone levels in plasma and nuclear thyroid hormone receptors in female rat liver, as well as to modify the effects of short-term whole body gamma-irradiation on nuclear thyroid-hormone receptors in the liver was evaluated after intraperitoneal administration of the drug at 0.4 mg/kg body weight. Ukrain had no effect on the concentration of thyroid hormones in rat blood and increased the concentration of thyroid hormone receptors in the liver of intact rats during the first 2 months after administration. Ukrain normalized the level of nuclear thyroid hormone receptors influenced by short-term whole body gamma-irradiation of rats with 1 Gy, beginning from the first day after administration of the drug. Thus, Ukrain can minimize the consequences of irradiation in the endocrine system of experimental animals.

Radiomodification effects of Ukrain, a cytostatic and immunomodulating drug, on intracellular glucocorticoid reception during short-term gamma-irradiation.

The ability of Ukrain, a cytostatic and immunomodulating semisynthetic compound of thiophosphate-modified alkaloids of Chelidonium majus L., to modify the effects of irradiation on intracellular glucocorticoid reception in female rat liver was evaluated after intraperitoneal administration of the drug at 0.4 mg/kg of body weight. Ukrain caused the normalization of the intracellular glucocorticoid-receptor system, influenced by short-term whole-body gamma-irradiation of rats with 1 Gy, beginning on the 10th day after administration of the drug. It was found that Ukrain minimized the consequences of irradiation in the endocrine system of the experimental animals.

Ukrain: a novel antitumor drug.

A review of the recent literature on the new anticancer drug Ukrain is provided herein. We review Ukrain, a thiophosphate derivative of alkaloids from Chelidonium majus L., its capacity to exert selective cytotoxic and cytostatic effects on tumor cells, simultaneously acting as an immune response modifier, its good tolerance and lack of side effects even after long-term application, perspectives of the application of this drug in oncology.

The influence of Ukrain on the growth of HA-1 tumor in mice: the role of cysteine proteinases as markers of tumor malignancy.

The influence of Ukrain (exerting a malignotoxic effect in vivo according to Nowicky J.W. et al [1]) on the growth of Hepatoma A (HA-1) tumors in mice and cysteine proteinases as markers of tumor growth and malignancy was studied. Ukrain administration (0.5 mg per mice of 20 g, in each of 5 i.p. injections) resulted in a reproducible and significant retardation of HA-1 tumor growth in the liver and a prolongation of lifespan compared with the untreated control. Repeated Ukrain administration to mice increased the number of macrophages in ascites, decreased the number of tumor cells, and concomitantly reduced the increased level of monocytes in peripheral blood. In ascitic cells the specific activity of cathepsin B was higher than in the intact liver and was not influenced by Ukrain; activity of cysteine proteinases studied in ascitic fluid was much higher than that in serum. Tumor growth was followed by a decrease in cathepsin B activity in the liver and serum at day 10. Ukrain administration has a tendency to normalize the disturbances of cathepsin B activity during tumor development. The cathepsin L activity changes in ascitic fluid were more impressive than those of cathepsin B, indicating the special role of this cysteine proteinase in HA-1 tumor growth and invasiveness. The role of cysteine proteinases as markers of tumor malignancy and invasiveness is discussed.

Induction of bimodal programmed cell death in malignant cells by the derivative Ukrain (NSC-631570).

Selective induction of malignant cell death is one of the major goals of effective and safe chemotherapy. Recent developments in the understanding of programmed cell death (PCD) or apoptosis are expected to provide new leads for a safer chemotherapy. The authors investigated whether the semisynthetic alkaloid thiophosphoric acid derivative Ukrain (NSC-631570) could induce PCD or apoptosis in human K562 leukaemia cells. Results showed that Ukrain induced two distinct modalities of cell death programmes. One modality corresponded morphologically to classical apoptosis or PCD characterized by blebbing and shedding of membrane vesicles with concomitant 51Cr release; however, the Ukrain-induced apoptosis was not associated with the characteristic nuclear DNA fragmentation. Higher concentrations of Ukrain induced a second cell death programme characterized by cell surface blister formation, high specific 51Cr release and extensive DNA polyploidy. These two cell death programmes are distinct from each other in that they are interphased by a silent period characterized by normal cell morphology and reduced specific 51Cr release.

Influence of Ukrain on DNA, RNA and protein synthesis in malignant cells.

3H labelled thymidine, uridine and leucine were used to evaluate the synthesis of DNA, RNA and proteins in malignant cells and normal cells incubated with Ukrain in different concentrations. Compared with the controls, the inhibiting effects of Ukrain are demonstrated in guinea pig hepatocytes, CIL hepatocytes, human tonsil cells, two murine lymphomas, murine myeloma, Yoshida cells, two HeLa strains, EsB- and EB- (murine) lymphomas. YAC-1, P815 and human WiDr cells. Ukrain inhibits the DNA, RNA and protein synthesis in malignant cell lines at relatively high concentrations and to a small extent in normal cells.

Influence of Ukrain on human xenografts in vitro.

Tumour cells were taken from human tumour xenografts (HTX) and serially transplanted into nude mice. These cells were used in a colony-forming assay in vitro. Tumour cells were incubated continuously for at least one week with several concentrations of the drug Ukrain. This was done with six different types and the colony formation was scored for each tumour. The drug effects were reported as percent T/C (Test/Control) for the numbers of colonies formed. Activity is considered to be present at T/C < 30%. A clear dose-response effect was found for Ukrain.

Mitogenic properties of Ukrain (NSC-6315170) on human peripheral blood monocytes: clinical implications.

Using a cell-proliferation assay, the authors examined and compared the mitogenic effects of Ukrain and phytohaemagglutinin (PHA) on human peripheral blood mononuclear cells (PBMCs), as well as their synergic effects. It was found that even a short period of pretreatment of the cell with Ukrain had a potent synergic effect on PHA mitogenesis resulting in significantly higher cell stimulation indices than those of PHA alone. Moreover, it was found that a short period of PHA treatment of the cells is almost imperative for Ukrain to exert its mitogenic effects. The mitogenic effect of Ukrain on human PBMCs is consistent with a previous clinical report which found that circulating lymphocytes were significantly increased in cancer patients treated with Ukrain. Thus the in vitro assay used in these studies may serve as a prognostic assay for potential patient responsiveness to Ukrain treatment, as well as a clinical parameter during Ukrain therapy.

Modulation of immune effector cell cytolytic activity and tumour growth inhibition in vivo by Ukrain (NSC 631570).

Ukrain is a semisynthetic compound consisting of alkaloids from Chelidonium majus L. conjugated to thiophosphoric acid, with immunomodulatory and therapeutic properties in cancer patients. The present in vitro studies demonstrate that Ukrain is an effective biological response modifier augmenting, by up to 48-fold, the lytic activity of splenic lymphocytes obtained from alloimmunized mice. The lytic activities of interleukin-2 (IL-2) treated spleen cells and peritoneal exudate lymphocytes were also significantly increased by the addition of Ukrain to the cell mediated lysis (CML) assay medium. The highest Ukrain-induced enhancement of splenic lymphocytolytic activity in vitro was found to occur at day 18 after alloimmunization was dose-dependent and specific for the immunizing P815 tumour cells. Since Ukrain was present only during the CML assays, its mode of action is thought to be via direct activation of the effector cells' lytic mechanism(s). The effect of Ukrain on the growth of Balb/c syngenic mammary adenocarcinoma was also evaluated. Intravenous, but not subcutaneous or intraperitoneal, administration of this drug was found to be effective in delaying tumour growth in an actual therapeutic protocol initiated five days after tumour implantation. No deleterious side-effects were observed using these in vivo treatment modalities. The role of macrophages in the observed retardation of tumour development was investigated, using peritoneal exudate macrophages (PEM) in cytotoxicity assays. Previous studies showed that PEM of mammary tumour-bearing mice lose their capacity to kill a variety of tumour target cells including the in vitro cultured homologous tumour cells (DA-3). Pretreatment of PEM from normal mice with 2.5 microM Ukrain for 24 h, followed by stimulation with either IFN-gamma or with lipopolysaccharide (LPS) plus IFN-gamma enhanced their cytotoxic activity. Treatment of PEM from tumour-bearing mice with 2.5 microM Ukrain and LPS results in a reversal of their defective cytotoxic response against DA-3 target cells. Furthermore, Ukrain alone, in the absence of a secondary signal, induced the activation of tumouricidal function of PEM from tumour-bearing, but not from normal, mice. These data indicate that Ukrain's in vivo effects against the development of mammary tumours may be due, at least in part, to its ability to restore macrophage cytolytic function.

Influence of Ukrain on patients with surgically treated breast cancer (introductory remarks).

Studies were undertaken to evaluate the influence of Ukrain on the postoperative period in patients with breast cancer. The group under Ukrain medication was comprised of 10 patients in stage T1-3NO-1MO. The control group was comprised of 22 patients in the same clinical and laboratory staging as those treated with Ukrain in the first year of postoperative follow-up metastatic lymph nodes were observed in one patient from the control group. It is planned to enlarge the groups and to continue the observation up to the third year to obtain reliable and more statistically significant data.

Influence of Ukrain on breast cancer.

The influence of Ukrain on breast cancer was studied in ten women and monitored in each patient by clinical examination, mammography, ultrasonography, histopathology and immunofluorescence in the tissue material obtained after radical mastectomy performed ten days after discontinuation of the treatment. Observations accumulated during the study and analytical data support the view that Ukrain, by changing the antigenic expression of malignant cells, make them recognizable for the immune system which rejects them in a host-versus graft manner.

Influence of Ukrain on patients with surgically treated breast cancer. Part I. Clinical and laboratory parameters.

Studies were undertaken to evaluate the influence of Ukrain on clinical and laboratory parameters in ten patients with breast cancer treated with the drug in the preoperative phase. The control group was composed of eight patients of similar age and advancement of the disease who did not receive Ukrain before mastectomy. Data from the present studies indicate that the drug is distinctly helpful from the surgical viewpoint by increasing tumour hardness and enlargement of metastatic lymph nodes. Furthermore, treatment with it is fully safe, with no side effects or allergic reactions.

Influence of Ukrain on patients with surgically treated breast cancer. Part II. Hormonal profile.

The present study was undertaken to evaluate the influence of Ukrain on the hormonal status of ten patients with breast cancer, treated in the preoperative phase by Ukrain. A control group consisted of eight patients of similar age and advancement of the disease, who did not receive the drug before mastectomy. The data were compared to those obtained in ten healthy women of similar age. The results indicate that Ukrain only slightly influences the hormonal dynamics and its destructive mode of action on neoplastic tissue does not significantly affect hormonal activities.

Influence of Ukrain on patients with surgically treated breast cancer. Part III. The immune system.

The present study was undertaken to evaluate the influence of Ukrain on immune parameters in ten patients with breast cancer, treated with the drug in the preoperative phase. The control group consisted of eight patients of similar age and advancement of the disease, who did not receive Ukrain before mastectomy. The data were compared with the parameters obtained in 15 healthy blood donors. It was found that treatment with Ukrain distinctly regulates cellular and, slightly, humoral immunity, non-specific immunity and also complement activity.

Influence of Ukrain on patients with surgically treated breast cancer. Part IV. Electromicroscopic and cytochemical evaluation.

The present studies were undertaken to evaluate by light and by electron microscopy the influence of Ukrain on the morphology of breast cancer. The studies were carried out on material obtained from ten patients with breast cancer, treated preoperatively with Ukrain. Control material for the studies was obtained from patients of similar age and advancement of the disease, who did not receive Ukrain. The data obtained in the present studies indicate that Ukrain is responsible for severe changes in morphology of tumour cells. Histological examination by both light and electron microscopy revealed cells characteristic of those undergoing apoptosis. The stromal changes of the tumour were characterized by intensive proliferation of the connective tissue accompanied by an immune reaction expressed by mononuclear infiltrates.