[Antitumor Activity of the Combination of Topotecan and Tyrosyl-DNA-Phosphodiesterase 1 Inhibitor on Model Krebs-2 Mouse Ascite Carcinoma].

Topotecan is a cytostatic drug from the camptothecin group, it acts by inhibiting topoisomerase 1 (TOP1). Tyrosyl-DNA phosphodiesterase 1 (TDP1) is capable of interfering with the action of TOP1 inhibitors, reducing their therapeutic efficacy. Suppression of TDP1 activity may enhance the effects of topotecan. In this work, we investigated the effect of the antitumor drug topotecan alone and in combination with a TDP1 inhibitor, a hydrazinothiazole derivative of usnic acid, on Krebs-2 mouse ascites tumors. We have previously shown that this derivative efficiently inhibits TDP1. In the present work, we show that both topotecan and the TDP1 inhibitor have an antitumor effect when evaluated separately. The combination of topotecan and the TDP1 inhibitor additively reduces both the weight of the ascites tumor and the number of cells in ascites. In mice, the TDP1 inhibitor alone or in combination with topotecan eliminated the tumor cells. After the combined intraperitoneal administration of these two compounds, we observed cells in which lipid droplets occupied almost the entire cytoplasm and the accumulation of cell detritus, which was absent in the samples collected from mice treated with each compound separately.

Precursors of thymic peptides as stress sensors.

INTRODUCTION: A large volume of data indicates that the known thymic hormones, thymulin, thymopoietin, thymosin-α, thymosin-ß, and thymic humoral factor-y2, exhibit different spectra of activities. Although large in volume, available data are rather fragmented, resulting in a lack of understanding of the role played by thymic hormones in immune homeostasis. AREA COVERED: Existing data compartmentalizes the effect of thymic peptides into 2 categories: influence on immune cells and interconnection with neuroendocrine systems. The current study draws attention to a third aspect of the thymic peptide effect that has not been clarified yet, wherein ubiquitous and highly abundant intranuclear precursors of so called 'thymic peptides' play a fundamental role in all somatic cells. EXPERT OPINION: Our analysis indicated that, under certain stress-related conditions, these precursors are cleaved to form immunologically active peptides that rapidly leave the nucleus and intracellular spaces, to send 'distress signals' to the immune system, thereby acting as stress sensors. We propose that these peptides may form a link between somatic cells and immune as well as neuroendocrine systems. This model may provide a better understanding of the mechanisms underlying immune homeostasis, leading thereby to the development of new therapeutic regimes utilizing the characteristics of thymic peptides.

Protective Effect of Peroxiredoxin 6 Against Toxic Effects of Glucose and Cytokines in Pancreatic RIN-m5F ß-Cells.

Taking into account a special role of pancreatic ß-cells in the development of diabetes mellitus, the effects of peroxiredoxin 6 (Prx6) on the viability and functional activity of rat insulinoma RIN-m5F ß-cells were studied under diabetes-simulating conditions. For this purpose, the cells were cultured at elevated glucose concentrations or in the presence of pro-inflammatory cytokines (TNF-α and IL-1) known for their special role in the cytotoxic autoimmune response in diabetes. It was found that the increased glucose concentration of 23-43 mM caused death of 20-60% ß-cells. Prx6 added to cells significantly reduced the level of reactive oxygen species and protected the RIN-m5F ß-cells from hyperglycemia, reducing the death of these cells by several fold. A measurement of insulin secretion by the RIN-m5F ß-cells showed a significant stimulatory effect of Prx6 on the insulin-producing activity of pancreatic ß-cells. It should be noted that the stimulatory activity of Prx6 was detected during culturing the cells under both normal and unfavorable conditions. The regulation of the NF-κB signaling cascade could be one of the mechanisms of Prx6 action on ß-cells, in particular, through activation of RelA/p65 phosphorylation at Ser536.

Immune response in the relapsing-remitting experimental autoimmune encephalomyelitis in mice: The role of the NF-κB signaling pathway.

Characteristics of the mouse model of relapsing-remitting experimental autoimmune encephalomyelitis (rEAE) closely resemble manifestations of multiple sclerosis in humans. In the present study, we investigated the mechanisms of inflammatory response, focusing on NF-κB pathway activation. Cytokine response in rEAE mice was multiphasic: the early phase was characterized by the increase in interferon-γ level in plasma. In the later stage, the level of interleukin-17, but not of interferon-γ, was increased. The early phase of rEAE was also accompanied by increased RelA/p65 phosphorylation at Ser276 in spleen cells, whereas the rEAE maintenance phase was characterized by RelA/p65 phosphorylation at Ser536 and IKK phosphorylation. The IKKα/ß inhibitor reduced interleukin-17 and interferon-γ levels in plasma and alleviated rEAE symptoms. The IKKα/ß inhibitor decreased IKK and p65(Ser536) phosphorylation, but doubled p65(Ser276) phosphorylation in rEAE mice. The increased RelA/p65(Ser276) phosphorylation coincided in time with the production of interferon-γ, Hsp72, and the early phase of IL-17 generation, whereas increased RelA/p65(Ser536) phosphorylation coincided with the activation of IKK, SAPK/JNK, and p53, as well as the late phase of IL-17 production, indicating the role of the RelA/p65 phosphorylation events in the induction and maintenance of rEAE.

The Role of p38 and CK2 Protein Kinases in the Response of RAW 264.7 Macrophages to Lipopolysaccharide.

The role of protein kinases p38 and CK2 (casein kinase II) in the response of RAW 264.7 macrophages to the lipopolysaccharide (LPS) from gram-negative bacteria was studied. Using specific p38 and CK2 inhibitors (p38 MAP kinase Inhibitor XI and casein kinase II Inhibitor III, respectively), we investigated the effects of these protein kinases on (i) LPS-induced activation of signaling pathways involving nuclear factor κB (NF-κB), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and interferon regulatory factor 3 (IRF3); (ii) expression of Toll-like receptor 4 (TLR4) and inducible heat-shock proteins HSP72 and HSP90; and (iii) production of interleukins IL-1α, IL-1ß, IL-6, tumor necrosis factor α, and IL-10. Activation of the proapoptotic signaling in the macrophages was evaluated from the ratio between the active and inactive caspase-3 forms and p53 phosphorylation. Six hours after LPS addition (2.5 µg/ml) to RAW 264.7 cells, activation of the TLR4 signaling pathways was observed that was accompanied by a significant increase in phosphorylation of IκB kinase α/ß, NF-κB (at both Ser536 and Ser276), p38, JNK, and IRF3. Other effects of macrophage incubation with LPS were an increase in the contents of TLR4, inducible heat-shock proteins (HSPs), and pro- and anti-inflammatory cytokines, as well as slight activation of the pro-apoptotic signaling in the cells. Using inhibitor analysis, we found that during the early response of macrophages to the LPS, both CK2 and p38 modulate activation of MAP kinase and NF-κB signaling pathways and p65 phosphorylation at Ser276/Ser536 and cause accumulation of HSP72, HSP90 and the LPS-recognizing receptor TLR4. Suppression of the p38 MAP kinase and CK2 activities by specific inhibitors (Inhibitor XI and Inhibitor III, respectively) resulted in the impairment of the macrophage effector function manifested as a decrease in the production of the early-response proinflammatory cytokines and disbalance between the pro- and anti-apoptotic signaling pathways leading presumably to apoptosis development. Taken together, our data indicate the inefficiency of therapeutic application of p38 and CK2 inhibitors during the early stages of inflammatory response.

The role of CK2 protein kinase in stress response of RAW 264.7 macrophages.

The role of casein kinase 2 (CK2) in the activation of the key NF-κB signaling cascade and other signaling and stress proteins during stress induced by exposure to nonthermal low-intensity electromagnetic radiation has been investigated. The ability of CK2 to regulate the activation of the NF-κB signaling cascade and upregulate the proinflammatory cytokine production and TLR4 receptor expression has been demonstrated. The existence of an alternative signaling pathway for NF-κB cascade activation directly involving protein kinase CK2 has been demonstrated using inhibition analysis in cells stressed by microwave irradiation.

Thymic peptides restrain the inflammatory response in mice with experimental autoimmune encephalomyelitis.

Modulation of autoimmune inflammation by the thymic peptides thymulin and thymopentin was studied in mice with acute experimental autoimmune encephalomyelitis (EAE), which resembles multiple sclerosis in humans. EAE was induced in NZW mice by a single immunisation with myelin basic protein coupled with adjuvants. Visible signs of pathology appeared on days 12-14 after the immunisation, peaked on days 20-25, were retained up to day 45, and then reverted. A biphasic cytokine response was also detected. In the "early" phase, which started at day 35, increased levels of interferon-gamma and interleukin-6 in the blood were observed; during the "delayed" phase, which started at day 48, the levels of plasma interleukin-17 and tumour necrosis factor-alpha were also raised. In addition, the phosphorylation of NF-kappaB signalling proteins and the production of heat shock protein Hsp72 were significantly increased in splenic lymphocytes from EAE-bearing mice. When applied intraperitoneally every other day for 30 days, either thymulin or thymopentin (15 µg per 100g of body weight) significantly reduced the disease severity compared to untreated EAE mice. The effect of thymulin but not thymopentin remained after its withdrawal. Thymulin reduced the cytokine response in both the early and the delayed phases, whereas thymopentin only reduced the "early phase cytokines" (IL-6 and interferon-gamma). Both peptides significantly reduced the level of phosphorylation of the NF-kappaB signalling protein IKK and the production of Hsp72 protein. The data presented here indicate the presence of time-dependent immune responses in EAE-bearing mice, which may be associated with the Th1 and Th17 subpopulations of T-cells. Thymulin and thymopentin demonstrated different patterns of activity, most likely via mechanisms involved in NF-kappa B signalling and Hsp72 expression.

[Effects of several inhibitors of intracellular signaling on production of cytokines and signal proteins in RAW 264.7 cells cultivated with low dose ammonium].

Effects of four inhibitors of NF-kappaB, SAPK/JNK and TLR4 signaling, namely, inhibitor XII, SP600125, CLI-095 and Oxpapc on a macrophage response to low dose ammonium were studied in RAW 264.7 cells. Low dose ammonium induced pro-inflammatory response in cells as judged from enhanced production of TNF-alpha, IF-gamma, and IL-6, and by activation of signal cascades. The increase in production of cytokines, namely TNF, IFN, and IL-6, demonstrated that low-dose ammonium induced a pro-inflammatory cellular response. In addition, an activation of NF-kappaB and SAPK/JNK cascades, as well as enhancement of TLR4 expression was shown. Each of used inhibitors reduced to a variable degree the pro-inflammatory response of RAW 264.7 cells on chemical toxin by decreasing cytokine production. The inhibitor of NF-kappaB cascade, IKK Inhibitor XII, was more effective, and not only prevented the development of pro-inflammatory response induced by ammonium, but also decreased cytokine production below control values. The inhibitor of extra cellular domains of TLR2 and TLR4 (OxPAPC) had almost the same anti-inflammatory effect, and an addition of the inhibitor of JNK cascade (SP600125) to cell culture practically neutralized effect of ammonium ions by decreasing cytokine production to control level. Inhibitory analysis showed that activation of RAW 264.7 cells induced by chemical toxin coincide incompletely with intracellular signaling pathways that were early determined regarding macrophage's response to toxin from gram-negative bacteria. Nevertheless, application of the inhibitors defended RAW 264.7 from toxic effect of the low dose ammonium.

[Slowing down the rate of irreversible age-related atrophy of the thymus gland by atopic autotransplantation of its tissue, subjected to long-term cryoconservation].

An experimental procedure has been developed enabling to slow down the rate of irreversible atrophy of the thymus gland. The atopic autotransplantation of its tissue subjected to prolonged cryoconservation enables one to inhibit the aging of the organism with respect to several biochemical and immunological indicators.

Role of heat shock protein hsp90 in formation of protective reactions in acute toxic stress.

The involvement of heat shock protein Hsp90 in pro-inflammatory response in male NMRI mice under conditions of acute toxic stress, caused by lipopolysaccharide from Gram negative bacteria, was studied using geldanamycin, a specific blocker of the activity of this protein. It is shown that the introduction of geldanamycin lowers total intoxication of the organism upon acute toxic stress caused by endotoxin. Thus, a decrease in cytokine TNF-alpha, IFN-gamma, IL-1, and IL-10 concentrations in blood serum of the geldanamycin-treated animals with acute toxic stress was found along with normalization of functional activity of nitric oxide producing peritoneal macrophages. Studying expression of receptor protein Tlr-4 as well of proteins of two signal cascades, NF-kappaB and SAPK/JNK, has shown that mechanisms of the geldanamycin protective effect are realized at the level of inhibition of Tlr-4 receptor expression, which provides for endotoxin-to-cell binding, and due to lowering the endotoxin-stimulated activation of signal cascades NF-kappaB and SAPK/JNK. The results suggest Hsp90 might be a therapeutic target in diseases accompanied by acute toxic stress.

[Stressful effects of chemical toxins at low concentrations].

Effects of three chemical compounds: ammonia, diethyl ether, and acetic acid, known as common environmental contaminants in technogenic accidents, were investigated in vivo and in vitro in low concentrations. When added in cultivation media, each of the chemicals has affected peritoneal macrophages and spleen lymphocytes isolated from male NMRI mice and led to a rise in the production of several cytokines, particularly the tumor necrosis factor-alpha and interferon-gamma, as well as the expression of the inducible form of heat shock proteins (HSP72 and HSP90-alpha) and in the activation of signal cascades NF-kappaB and SAPK/JNK. The increase of the nitric oxide (NO) production in macrophages has been observed only when ammonia was added in cultivation media. Also, low concentrations of all compounds investigated led to the activation of the expression of receptor protein TLR4. When mice were exposed to airborne toxic contaminants in a hermetically sealed experimental chamber, an increase in the concentrations of cytokines, heat shock proteins, and signal proteins in immune cells was also observed in response to low concentrations of all chemicals investigated. Similarly to in vitro experiments, the NO production was augmented only in the presence of the airborne ammonia. The results indicate the environmental hazard of chemical contaminants even in rather low concentrations, which nevertheless lead to the stress response.

[The role of protein kinase SAPK/JNK in cell responses to low-intensity nonionizing radiation].

The effect of low-intensity lases light (0.2 mW/cm2, 632.8 nm, exposure time 1 min) or centimeter waves (8.15-18 GHz, 1 W/cm2, exposure time 1 h) on PhosphoSAPK/JNK production in mice lymphocytes was investigated. Normal isolated spleen lymphocytes or cells incubated previously with geldanamycin, an inhibitor of heat shock protein 90 (HSP90), were used in the experiments. A significant stimulation of PhosphoSAPK/JNK production in lymphocytes after treatment with laser light or microwaves has been shown in both cell models. It was proposed that the activation of SAPK/JNK signal pathway plays one of the central roles in cellular stress response to low-power nonionizing radiation.

[Immunomodulatory effects of thymopentin under acute and chronic inflammations in mice].

The effects of thymopentin, a synthetic analogue of the active center of the thymus hormone thymopoietin, on the immune status of mice with two different models of inflammation induced by injection of lipopolysaccaride (LPS) from gram-negative bacteria were studied. Acute inflammation was induced by a single injection of LPS in a dose of 250 microg/100 g of body weight, and chronic inflammation (sepsis) was modeled by a daily injection of LPS for 11 days with a gradual increase in the dose range from 25 to 250 microg/100 g of body weight. Under acute inflammation, a preliminary injection of thymopentin did not induce any additional stimulation in cytokine production increased by LPS. On the contrary, whereas the chronic introduction of LPS was characterized by a depressed production of several cytokines, thymopentin produced an immunostimulating effect. Thus, an increase in the production of nitric oxide, interferon-gamma, and Hsp70 was demonstrated. In addition, a more effective restoration of the number of thymus cells, as well as an increase in macrophage tumor necrosis factor-alpha production were observed after repeal of LPS + hormone injections. The results show that preliminary application of thymopentin promotes the regulation of immune cell activity under acute and chronic inflammation.

[Effect of geldanamycin on the expression of signal proteins and heat shock proteins in normal mice lymphocytes].

The effects of geldanamycin, which is known as inhibitor of heat shock protein 90 activities, on expression of several signal and heat shock proteins were studied by Western blot analysis in cultivated spleen lymphocytes isolated from male NMRI mice. It has been revealed that cultivating the cells with geldanamycin resulted in decrease in transcription factor NF-kappaB amount, as well as decrease in its phosphorylated form, pNF-kappaB, and lowering in its suppressor, IkappaB-alpha. Besides, cells cultivated with geldanamycin demonstrated significant decrease in the amount of protein kinase SAPK(JNK). The modifications in signal pathways, which had been induced by geldanamycin, pointed to direct influence of the antibiotics on cellular stress response to damaging impact. This assumption was examined with the model of cellular stress response induced by low-level laser radiation. It was proved that Hsp90-binding drug, geldanamycin, significantly decreased in vitro stress response to laser light via lowering the production of heat shock proteins, Hsp70 and Hsp25, both in irradiated lymphocytes and in theirs intracellular structures. These findings show the prospect for using of geldanamycin in various therapies that are compromised with objectionable side effects manifested as heightened stress response in immune cells.

[The role of heat shock proteins HSP90 in the response of immune cells to centimeter microwaves].

The effects of low-level electromagnetic waves (8.15-18 GHz, 1 microW/cm2, 1 h) on the production of heat shock proteins, several cytokines, and nitric oxide in isolated mouse macrophages and lymphocytes were examined both under normal conditions and after the treatment of the cells with geldanamycin (GA), a depressor of activity of the heat shock protein 90 (Hsp90). The irradiation of cells without GA induced the production of Hsp70, nitric oxide (NO), interleukin-1beta (IL-1beta), interleukin-10 (IL-10), and the tumor necrosis factor -alpha (TNF-alpha). No changes in the production of Hsp90 in irradiated cells were observed, but intracellular locations of Hsp25 and Hsp70 altered. The preliminary treatment of cells with GA did not remove the effects of microwaves: in these conditions, the synthesis of all cytokines tested, nitric oxide, as well as total and membrane amount of Hsp70, and the amount of Hsp25 in the cytoplasm and cytoskeleton increased. Moreover, the exposure of cells incubated with GA resulted in the reduction of Hsp90-alpha production.

[Atopically transplanted thymus tissues are capable of distantly changing the metabolic status of the body].

It has been found that the atopic transplantation of the thymus tissue from young animals in different variants is capable of significantly decreasing the rate of irreversible age-related atrophy of the thymus, which retards the age-dependent degradation of the T-cell unit of the immune system of the organism.

[Effects of centimeter waves on the immune system of mice in endotoxic shock].

The effects of centimeter waves (8.15-18 GHz, 1 microW/cm2, 1 h daily for 10 days; MW) on the production of the tumor necrosis factor alpha, interleukin-lalpha, interleukin-1beta, interleukin-2, and the expression of interleukin-6, interleukin-10, interferon-gamma, nitric oxide and HSP27, HSP72 and HSP90alpha in mice irradiated before or after LPS injection were studied. An acute endotoxic model was produced by a single LPS injection. The effects of microwaves on nitric oxide, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma were dependent on the functional status of exposed animals. Thus, an exposure of healthy mice to microwaves for 10 days was followed by a decrease in nitric oxide and interferon-gamma production, and an increase in the production of the tumor necrosis factor-alpha and interleukin-6. On the contrary, an exposure to MW before intoxication resulted in an increase in the synthesis of nitric oxide and interferon-gamma as well as a decrease in the concentration of the tumor necrosis factor-alpha and interleukin-6 in blood of mice in endotoxic shock. When microwave exposure was used after LPS injection, it did not provide any protective effect, and preliminary irradiation enhanced the resistance of the organism to endotoxic shock.

[Protective effect of low-power laser radiation in acute toxic stress].

The effect of preliminary short-term irradiation with He-Ne laser light (632.8 nm, 0.2 mW/cm2) of the thymus zone projection of male NMRI mice subjected to acute toxic stress on the responses of immune cells was studied. Stress was modeled by lipopolysaccharide injection, 250 mg/100 g of body weight, which induced a significant increase in the production of several macrophage cytokines, IL-1alpha, IL-1beta, IL-6, IL-10 and TNF-alpha. A single irradiation with laser light did not provoke considerable variations in NO production in cells but induced an enhancement in the production of heat shock proteins Hsp25, Hsp70, and Hsp90. Nevertheless, when irradiation with red laser light was applied prior to toxic stress, considerable normalization of production of nearly all cytokines studied and nitric oxide was observed. Moreover, the normalization of production of heat shock proteins has been shown in these conditions. Thus, preliminary exposure of a small area of animal skin surface provoked a significant lowering in the toxic effect of lipopolysaccharide.

[Effect of low-intensity laser radiation (632.8 nm) on immune cells isolated from mice].

The effect of in vitro exposure to low-power laser light with a power density of 0.2 mW/cm2 and a wavelength of 632.8 nm induced by helium-neon laser on the functional activity of macrophages and splenic lymphocytes was studied. If the exposure period did not exceed 60 sec, the stimulation in interleukin-2 (IL-2) and nitric oxide (NO) production, as well as an increase in the activity of natural killer cells were observed. The increase of irradiation dose by prolongation of the exposure duration up to 180 s induced a significant decrease in NO production and natural killer cell activity, but IL-2 production was not different from the control level. A remarkable decrease in interferon-gamma (IFN-gamma) production was observed following laser light exposure of cells for 60 or 180 s, whereas under lower doses (exposure for 5 or 30 sec) IFN-gamma production increased. Irradiation of isolated macrophages induced a significant stimulation of cellular tumor necrosis factor-alpha (TNF- alpha) production at all dboes used, and, what is more important, an enhancement in both TNF-a phaand interleukin-6 (IL-6) production was revealed as early as after a 5-s exposure. In this case, more prolonged exposure periods, 60 and 180 s, either did not induce changes in IL-6 production (in macrophages) or decreased IL-6 production (in lymphocytes). Thus, upon in vitro exposure of cells to extremely low-power laser light, a basic tendency was observed: short-term irradiation predominantly induced stimulation in secretory activity of cells, whereas prolongation of exposure mainly induced immunosuppression. The only exception to the rule was a change in interleukin-3 (IL-3) production, which decreased after short-time exposure, but, on the opposite, increased when the cells were exposed for 180 s. In addition, a high sensitivity to extremely low-power laser light was supported by expression of the inducible heat shock protein, Hsp70, the effect being observed at all doses used, including the exposure for 5 s. At the same time, expression of another heat shock protein, Hsp90, was somewhat reduced after irradiation of cells with laser light.