Pharmacological study on kefir--a fermented milk product in Caucasus. I. On antitumor activity (1)]

The antitumor activity of kefir (YK-1), a fermented milk product in Caucasus, was investigated. YK-1 at oral doses of 100 or 500 mg/kg inhibited the proliferation of solid tumor of Ehrlich ascites carcinoma transplanted subcutaneously in mice. YK-1 did not show an inhibitory effect on the ear swelling induced contact dermatitis caused by picryl chloride (PC-CD). However, YK-1 inhibited the immunosuppression in Ehrlich carcinoma-bearing mice and with the frozen and dried ascites of the tumor-bearing mice containing immunosuppressive substances (EC-sup) in PC-CD-induced mice. And also, YK-1 activated the immunosuppressive activity of spleen cells of mouse treated with EC-sup. These results suggest that YK-1 may have antitumor activity against Ehrlich carcinoma and activate the immunosuppression with it.     

端粒重复扩增技术测定端粒酶在不同肿瘤细胞中的分布

目的：确定端粒酶在几种恶性肿瘤细胞和肿瘤组织中的分布状态及其临床意义。方法：采用端粒重复扩增技术（Telomere Repeat Amplifecation Protocol,TRAP）测定人鼻咽癌细胞KB、新鲜乳癌组织细胞、乳癌转移淋巴结细胞、人宫颈癌细胞系Hela、小鼠腹水癌细胞P388、人白血病细胞K652及人肺癌细胞A549中端粒酶的表达水平。结果：测试显示,新鲜乳癌组织细胞、人宫颈癌细胞Hela、小鼠淋巴细胞白血病细胞系P388及人肺癌细胞系A549均出现端粒酶的阳性表达,阳性率为50%。结论：证实了端粒酶在一些恶性肿瘤细胞组织中的存在,同时也反映了端粒酶与上述肿瘤细胞的恶性增殖行为的密切联系。     

The effects of fractions (chalones) obtained from lymphoid organs on the immune response in vivo.

Aqueous extracts of calf and pig lymphoid organs were prepared and fractionated by means of gel filtration, ion exchange chromatography, and isoelectric focusing. These fractions, which had been previously assessed on mitogen-stimulated mouse spleen lymphocytes and other cells in vitro, were tested for their in vivo activity on humoral (haemolytic PFC in mice) and on cell-mediated immunity (skin allograft survival in mice, lymph node weight assay in rats, and systemic GvH-reaction in mice). None of these several fractions elicited either biologically significant or reproducible inhibitory effects. In particular, two fractions, a high and a small molecular weight fraction which were strongly inhibitory in vitro, remained without any chalone-like activity in these in vivo assays. Our results therefore failed to support the existence of a lymphocyte chalone.     

A role for calcium-activated calmodulin in murine nonspecific cell-mediated cytotoxicity

The role of Ca++ in mouse nonspecific cell-mediated cytotoxicity was studied using the calcium ionophore A23187, inhibitors of calmodulin activity, and agents which modulate cyclic AMP concentration. A23187 markedly enhanced spleen lymphocyte cytotoxicity against SV3T3 target cells, suggesting that Ca++ influx enhances cytolytic activity. This conclusion was supported by experiments in which verapamil, a Ca++ channel blocker, inhibited normal and ionophore-induced cytotoxicity. A23187 also enhanced cytolysis of YAC-1 cells in a 16-hr 51Cr release assay, but had little effect in the more typical 4-hr assay. Lysis of BHK, a cell line resistant to murine natural cytotoxicity, could not be induced by A23187. However, hamster effector cells, which can lyse xenogeneic target cells, showed increased against either SV3T3 or BHK. This indicates that the effect of the ionophore was not due to nonspecific release of toxic products from the effector cells. Both normal and ionophore-enhanced lysis were also inhibited by the phenothiazines chlorpromazine and trifluoperazine, which block the activity of calmodulin. A more specific calmodulin activity inhibitor, W13, was also shown to profoundly inhibit cytotoxicity induced by A23187. These results suggest that Ca++ acts as a stimulus-response coupler in cell-mediated cytotoxicity, and that calmodulin mediates the effects of the Ca++. Furthermore, cyclic AMP appears to modulate the action of Ca++ since agents which increase cAMP levels reduce the effects of A23187.     

The role of NK cells in antitumor activity of dietary fucoidan from Undaria pinnatifida sporophylls (Mekabu)

Fucoidan from Mekabu (sporophyll of Undaria pinnatifida), a dietary alga, exerts antitumor activity possibly through enhancing the immune response. The present report describes the effects of dietary Mekabu fucoidan on the tumor growth of mouse A20 leukemia cells and on T cell-mediated immune responses in T cell receptor transgenic (DO-11 - 10 - Tg) mice. The animals were fed with a diet containing 1% Mekabu fucoidan (0.034 +/- 0.003 g/mouse/day) for 10 days and subcutaneously (s. c.) inoculated with A20 leukemia cells. Thereafter, the mice were fed with the diet containing fucoidan for 40 days. Mekabu fucoidan inhibited tumors by 65.4 %. We studied how the killer activities of T cell-mediated and natural killer (NK) cells are augmented in DO-11 - 10 mice fed with Mekabu fucoidan. The cytolytic activities of ovalbumin (OVA), which is specific against OVA-transfected A20 (OVA-A20) B lymphoma cells, and NK cells against YAC-1 were significantly enhanced in the mice fed with fucoidan compared with a basic diet. Thus, these findings suggested that Mekabu fucoidan mediates tumor destruction through Th1 cell and NK cell responses.     

白细胞介素12的生物学功能与药理学作用

白细胞介素12(IL-12)是由巨噬细胞等抗原提呈细胞产生的细胞因子。IL-12对T细胞、自然杀伤细胞具有免疫调节作用,促进Th1介导的细胞免疫反应。研究证实,IL-12对小鼠的肿瘤和各种感染性疾病等有治疗作用。国内外学者采用不同的治疗途径及不同的联合方案进行了大量的尝试。本文对目前IL-12的生物学功能及药理学作用作一综述。     

荔枝核的抑瘤作用及对肝癌组织端粒酶活性的影响

目的:建立肝癌荷瘤小鼠模型,研究荔枝核水提物对小鼠模型实体瘤生长的影响。方法:小鼠S180肿瘤细胞与肝癌细胞混悬液用生理盐水按1∶1进行稀释,制成含瘤腹水混悬液;在给药前24h每只小鼠腋窝皮下接种0.2mL,整个操作在无菌条件下60min内完成。对小鼠S180肿瘤细胞、肝癌细胞连续10d给予荔枝核水提物,观察S180肿瘤细胞和肝癌细胞的生长情况、肝癌荷瘤小鼠体内Bcl -2的水平及肝癌细胞的凋亡情况。结果:荔枝核水提物能显著抑制S180肿瘤细胞和肝癌细胞的生长(P<0.01) ;荔枝核能显著升高肝癌荷瘤小鼠体内Bcl -2的水平(P<0.05或P<0.01) ;荔枝核能够显著促进肝癌细胞的凋亡。结论:荔枝核的抑瘤作用可能与升高动物体内的Bcl -2水平及促进癌细胞的凋亡有关。     

Influence of lead acetate on hypersensitivity. Experimental study

Recent studies showed that lead acetate has an important immunotoxicity for the phagocytic activity as well as humoral and cell-mediated immunity. We studied the influence of lead acetate on immediate and delayed hypersensitivity. The lead acetate exerts an important action on hypersensitivity reactions whether on rat mast cells degranulation (immediate hypersensitivity) or on contact hypersensitivity.     

Inhibition of cytotoxicity by sulfasalazine. I. Sulfasalazine inhibits spontaneous cell-mediated cytotoxicity by peripheral blood and intestinal mononuclear cells from control and inflammatory bowel disease patients

We have studied the effects of sulfasalazine and its metabolites on cell-mediated cytotoxicity by peripheral blood and intestinal mononuclear cells from both control and inflammatory bowel disease (IBD) patients. Sulfasalazine and sulfapyridine, as well as hydrocortisone and nordihydroguaiaretic acid inhibited spontaneous cell-mediated cytotoxicity by control and IBD peripheral blood cells. Sulfasalazine and nordihydroguaiaretic acid inhibited spontaneous cell-mediated cytotoxicity by control and IBD intestinal mononuclear cells cultured for 72 h in media alone. In contrast, 5-aminosalicylate, indomethacin and benzylimidazole had no effect on cytotoxicity by any cell population. Lectin-induced, antibody-dependent and interleukin-2-induced cell-mediated cytotoxicity, as well as lymphokine-activated killing were not inhibited by the drugs: inhibitory effects in these assays were primarily upon the underlying spontaneous cell-mediated cytotoxicity. The inhibition induced by sulfasalazine, sulfapyridine and nordihydroguaiaretic acid could not be reversed by adding the lipoxygenase metabolites leukotriene B4 or 12-hydroxyeicosatetraenoic acid. These findings demonstrate that spontaneous cell-mediated cytotoxicity by control and IBD mononuclear cells can be inhibited by sulfasalazine.     

Stress and immune responses. III. Effect of restraint stress on delayed type hypersensitivity (DTH) response, natural killer (NK) activity and phagocytosis in mice

Several experiments were conducted to evaluate the influences of restraint stress on cell-mediated immune events in mice. Delayed type hypersensitivity response to sheep red blood cells was inhibited by the stress, regardless of the timing of restraint stress loading. The activity of phagocytosis of macrophages in vitro and in vivo were measured by using the zymosan-particle uptake method and the carbon clearance test, respectively. Both activities were decreased in restraint-stressed mice. The suppressed carbon clearance rate in stressed mice, however, was recovered by the transfusion of serum from normal mice. Natural killer activity in spleen cells was decreased to 30-50% of the control in stressed mice. However, no suppressor cells which could inhibit NK activity existed in the spleen from stressed mice. These results show that the restraint stress suppresses various kinds of cell-mediated immune events, which might play an important role in anti-tumor immunity.     

Development of a flow cytometry assay for the identification and differentiation of chemicals with the potential to elicit irritation, IgE-mediated, or T cell-mediated hypersensitivity responses.

These studies were conducted to investigate the potential use of a flow cytometric analysis method for the identification and differentiation of chemicals with the capacity to induce irritation, IgE- or T cell-mediated hypersensitivity responses. An initial study investigated the ability of equally sensitizing concentrations (determined by local lymph node assay) of IgE-mediated (Toluene Diisocyanate-TDI) and T cell-mediated (Dinitrofluorobenzene-DNFB) allergens to differentially modulate the IgE+B220+ population in the lymph nodes draining the dermal exposure site. Sodium lauryl sulfate (SLS) was also tested as a nonsensitizing irritant control. Female B6C3F1 mice were dermally exposed once daily for 4 consecutive days, with the optimum time point for analysis determined by examining the IgE+B220+ population 8, 10, and 12 days post-initial chemical exposure. At the peak time point, day 10, the IgE+B220+ population was significantly elevated in TDI (41%), while moderately elevated in DNFB (18%) exposed animals when compared to the vehicle (0.8%), and remained unchanged in SLS (2.2%) exposed animals when compared to the ethanol control (2.5%). Experiments in our laboratory and others have demonstrated that the draining lymph node B220+ population becomes significantly elevated following exposure to allergens (IgE- and T cell-mediated), not irritants, allowing for their differentiation. An existing mouse ear swelling assay was used to identify chemical irritants. Therefore, using the endpoints of percent ear swelling, percent B220+ cells, and percent IgE+B220+ cells, a combined irritancy/phenotypic analysis assay was developed and tested with tetradecane (irritant), toluene diisocyanate, trimellitic anhydride (IgE-mediated allergens), benzalkonium chloride, dinitrofluorobenzene, oxazolone, and dinitrochlorobenzene (T cell-mediated allergens) over a range of concentrations. Based upon the pattern of response observed, a paradigm was developed for continued evaluation: Irritant exposure will result in significant ear swelling without altering the B220+ or IgE+B220+ populations. Exposure to sensitizers (IgE-mediated or T cell-mediated) will increase the B220+ population and the percent ear swelling will remain unchanged or will significantly increase, depending on the irritancy capacity of the chemical. Both the IgE+B220+ and B220+ populations will become elevated at the same test concentration following exposure to IgE-mediated, hypersensitivity inducing allergens. At its peak, the percent of IgE+B220+ cells will be equal to the percent of B220+ cells. The B220+ population will increase at a lower test concentration than the IgE+B220+ population, following exposure to T cell-mediated, hypersensitivity inducing allergens. At its peak, the percent of IgE+B220+ cells will reach less than half that of the percent of B220+ cells. The irritancy/phenotypic analysis method may represent a single murine assay able to identify and differentiate chemicals with the capacity to induce irritation, or IgE-mediated or T cell-mediated responses.     

Neutralization of complement regulatory proteins augments lysis of breast carcinoma cells targeted with rhumAb anti-HER2.

The capacity of recombinant human monoclonal anti-p185HER2 IgG (rhumAb anti-HER2) to activate human complement was investigated. Complement activation by rhumAb anti-HER2 on various human breast carcinoma cell lines resulted in deposition of complement proteins on these cells. Complement activation was also observed in a solid-phase binding assay, in which purified p185HER2 was immobilized onto a microtiter plate. rhumAb anti-HER2 induced some complement-mediated tumor cell lysis by rabbit complement, but not by human complement. Analysis of membrane complement regulatory proteins (mCRP) on breast carcinoma cells revealed a heterogenous expression of CD46, CD55 and CD59. After blocking the mCRP activity with specific antibodies, rhumAb anti-HER2 induced about 15% lysis of p185HER2-expressing tumor cells. Tumor cell sensitization with rabbit polyclonal anti-tumor antiserum following mCRP neutralization, augmented cell lysis from 10 to 80%. Expression of mCRP was upregulated by treatment with PMA, and correlated with increased protection of the tumor cells from complement lysis. These results suggest that humanized antibodies like rhumAb anti-HER2 promote complement activation leading to tumor cell phagocytosis and cell-mediated cytotoxicity. They further demonstrate that a successful tumor immunotherapeutical approach, based on antibody and complement treatment, requires mCRP neutralization.     

Inhibitory effects of green tea and grape juice on the phenol sulfotransferase activity of mouse intestines and human colon carcinoma cell line, Caco-2

Tea and fruit juices are beverages consumed daily all over the world. The present study reports the inhibitory effects of these beverages on the activity of mammalian intestinal phenol sulfotransferases (P-STs). Green tea strongly inhibited the E. coli-expressed mouse intestinal P-ST activity in vitro. (-)-Epigallocatechin gallate (EGCG) was found to be the most potent inhibitor among the catechins tested (IC50=0.93 microM). (-)EGCG also inhibited the P-ST activity of the human colon carcinoma cell line, Caco-2. Kinetic analysis showed that the inhibition was competitive. Among fruit juices examined (apple, grape, grapefruit and orange), grape juice exhibited the most potent inhibitory action on the P-ST activity of mouse intestines and human colon carcinoma cells. The inhibitory activity of grape juice was located mainly in the skin and seeds. Flavonols, such as quercetin and kaempferol, inhibited the P-ST activity at low concentrations. These observations suggest the possible inhibition of P-ST activity in human intestines by green tea or grape juice.     

Manganese chloride enhances natural cell-mediated immune effector cell function: effects on macrophages

A single intramuscular injection of MnC12 in mice caused an increase in macrophage functional activity. Spleen cell antibody-dependent cell-mediated cytotoxicity against both chicken erythrocytes and P815 tumor cell targets was enhanced 24 h following a single injection of MnC12. Enhanced antibody-dependent cell-mediated cytotoxicity activity following MnC12 treatment was not associated with a change in spleen cellularities compared with saline-injected mice. Resident peritoneal macrophages from mice injected intramuscularly with MnC12 displayed enhanced phagocytic activity for chicken erythrocytes in the presence or absence of opsonizing antibody. Enhanced cytolytic activity against P815 mastocytoma target cells and enhanced cytostatic activity against MBL-2 lymphoma target cells was also observed for nonelicited resident peritoneal macrophages from mice injected intramuscularly with MnC12. There were no differences in the cellularity or relative number of adherent cells obtained from the peritoneal cavity of saline or MnC12-injected mice. These enhanced macrophage functions were associated with the induction of increased interferon levels in mice injected with MnC12.     

RS034对免疫抑制小鼠的抗癌活性

采用 Levey & Medawar 的两次免疫程序,制备了兔抗小鼠胸腺细胞血清(RAMTS),并用以建立小鼠的细胞免疫抑制模型。使用这个模型研究了7β-羟基胆固醇双琥珀酸单酯钠(RS034)的抗肿瘤活性。结果表明细胞介导免疫抑制并没有显著影响 RS034的抗肿瘤活性(P>0.05)。因此推测 RS034的抗肿瘤活性可能是与机体免疫状态关系不大的一种直接作用。     

In vitro activity of a Solanum tuberosum extract against mammary carcinoma cells

We investigated the antitumor properties of a Solanum tuberosum extract (STE) on F3II mouse mammary carcinoma cells. STE significantly inhibited adhesion on fibronectin-coated surfaces and blocked migration of tumor cells in vitro. A major gelatinolytic activity (gelatinase) of 82 kD was identified in STE by zymographic analysis and characterized by exposure to different experimental conditions. Proteolytic activity of STE may be responsible, at least in part, for the in vitro effects on mammary carcinoma cells.     

Novel 6-substituted uracil analogs as inhibitors of the angiogenic actions of thymidine phosphorylase.

Thymidine phosphorylase (TP) catalyzes the reversible phosphorolysis of thymidine and other pyrimidine 2'-deoxyribonucleosides. In addition, TP has been shown to possess angiogenic activity in a number of in vitro and in vivo assays, and its angiogenic activity has been linked to its catalytic activity. A series of 5- and 6-substituted uracil derivatives were synthesized and evaluated for their abilities to inhibit TP activity. Among the most active compounds was a 6-amino-substituted uracil analog, 6-(2-aminoethyl)amino-5-chlorouracil (AEAC), which was a competitive inhibitor with a K(i) of 165 nM. The inhibitory activity of AEAC was selective for TP, as it did not inhibit purine nucleoside phosphorylase or uridine phosphorylase at concentrations up to 1 mM. Human recombinant TP induced human umbilical vein endothelial cell (HUVEC) migration in a modified Boyden chamber assay in vitro, and this action could be abrogated by the TP inhibitors. The actions of the inhibitors were specific for TP, as they had no effect on the chemotactic actions of vascular endothelial growth factor (VEGF). HUVEC migration was also induced when TP-transfected human colon and breast carcinoma cells were co-cultured in the Boyden chamber assay in place of the purified angiogenic factors, and a TP inhibitor blocked the tumor cell-mediated migration almost completely. These studies suggest that inhibitors of TP may be useful in pathological conditions that are dependent upon TP-driven angiogenesis.     

Adenosine stimulation of the proliferation of colorectal carcinoma cell lines. Roles of cell density and adenosine metabolism

Adenosine is a purine nucleoside which is present at micromolar concentrations in the extracellular fluid of solid cancers as a result of tissue hypoxia. Adenosine acts to promote tumor survival by inhibiting the cell-mediated anti-tumor immune response. However, its role in modulating proliferation of the tumor cell population is unclear. Differing results have been obtained using adenosine analogues or by interfering with adenosine metabolism. We examined the effect of adenosine itself on DNA synthesis and cell growth in six different human and mouse colorectal carcinoma cell lines, from different sites and at different stages of differentiation. Adenosine given as a single dose consistently stimulated DNA synthesis and cell proliferation in all cell lines tested, with an EC(50) of 3.8-30 microM and a maximum stimulation being reached at 10-100 microM. AMP and ATP also stimulated cell proliferation at similar doses. The stimulation by adenosine varied depending upon the culture cell density, with the greatest mitogenic effect at subconfluent densities. Adenosine was metabolized by cellular adenosine deaminase and adenosine kinase. The half-life (t(1/2)) for the decline in adenosine concentration in the medium following a single addition was between 40 min and 3 hr depending on the cell line and culture conditions. The rate of production of endogenous adenosine was low under normoxic culture conditions. Continuous dosing of cultures with adenosine to provide a steady-state concentration showed that proliferation could be stimulated by low micromolar concentrations of adenosine. We conclude that adenosine is stimulatory to the growth of human colorectal carcinoma cells at concentrations present within the tumor extracellular environment.     

Arugomycin, a new anthracycline antibiotic. III. Biological activities of arugomycin and its analogues obtained by chemical degradation and modification

Biological activities of arugomycin and its analogues obtained by chemical degradation and modification were evaluated. Differences in the sugar moieties affected their biological activities including induction of differentiation of mouse Friend erythroleukemia cells and mouse myeloid leukemia cells, antitumor activities against sarcoma S-180, Ehrlich ascites carcinoma and P388 leukemia, and cytotoxicity against murine leukemia cells. Some relationships were found between the sugar moieties and biological activities.     

Inhibition of mammalian tumour thymidylate synthetase by 2'-deoxyuridine 5'-phosphate analogues

This paper presents improved synthetic methods for the modification of 2'-deoxyuridine-5'-monophosphate and its 5-fluoro derivative, using trimethylphosphate in aqueous medium at pH 10. These modifications include methylation of the pyrimidine ring N(3) and/or esterification of the phosphate group. The 5'-methyl ester of dUMP was neither a substrate nor an inhibitor of Ehrlich ascites carcinoma thymidylate synthetase. By contrast, the corresponding methyl ester of FdUMP was a tight-binding inhibitor of the enzyme from L1210, Ehrlich ascites carcinoma and CCRF-CEM cells. 3-Methyl-dUMP, fixed in the 4-keto form, exhibited only very weak substrate activity with the Ehrlich ascites carcinoma enzyme. The dUMP analogues 5-ethyl-dUMP and 5-propyl-dUMP were found to be competitive inhibitors of thymidylate synthetase from L1210, Ehrlich ascites carcinoma and HeLa cells, the former being the more potent inhibitor. Both analogues were shown to bind cooperatively to each of the mouse tumour enzymes. Two molecules of inhibitor interacted with a single enzyme molecule, reflected by the parabolic character of the replots of the slope versus inhibitor concentration. The parent dTMP was a stronger inhibitor of the mouse tumour enzymes than its higher alkyl homologues.