Cancer cell iron metabolism and the development of potent iron chelators as anti-tumour agents

Cancer contributes to 50% of deaths worldwide and new anti-tumour therapeutics with novel mechanisms of actions are essential to develop. Metabolic inhibitors represent an important class of anti-tumour agents and for many years, agents targeting the nutrient folate were developed for the treatment of cancer. This is because of the critical need of this factor for DNA synthesis. Similarly to folate, Fe is an essential cellular nutrient that is critical for DNA synthesis. However, in contrast to folate, there has been limited effort applied to specifically design and develop Fe chelators for the treatment of cancer. Recently, investigations have led to the generation of novel di-2-pyridylketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) group of ligands that demonstrate marked and selective anti-tumour activity in vitro and also in vivo against a wide spectrum of tumours. Indeed, administration of these compounds to mice did not induce whole body Fe-depletion or disturbances in haematological or biochemical indices due to the very low doses required. The mechanism of action of these ligands includes alterations in expression of molecules involved in cell cycle control and metastasis suppression, as well as the generation of redox-active Fe complexes. This review examines the alterations in Fe metabolism in tumour cells and the systematic development of novel aroylhydrazone and thiosemicarbazone Fe chelators for cancer treatment.     

In vitro inhibition of bacterial growth by iron chelators

The antimicrobial activity of the iron(III)-selective 3-hydroxypyridin-4-one chelators, CP251(1) and CP252(2), was evaluated in comparison with that of diethylenetriamine-penta acetic acid (3). CP251 was found to exhibit an inhibitory effect on the growth of both Gram-positive and Gram-negative bacteria. CP251 may find application in the treatment of external infections such as those associated with wounds.     

Prevention of oxidant-induced cell death by lysosomotropic iron chelators

Intralysosomal iron powerfully synergizes oxidant-induced cellular damage. The iron chelator, desferrioxamine (DFO), protects cultured cells against oxidant challenge but pharmacologically effective concentrations of this drug cannot readily be achieved in vivo. DFO localizes almost exclusively within the lysosomes following endocytic uptake, suggesting that truly lysosomotropic chelators might be even more effective. We hypothesized that an amine derivative of alpha-lipoamide (LM), 5-[1,2] dithiolan-3-yl-pentanoic acid (2-dimethylamino-ethyl)-amide (alpha-lipoic acid-plus [LAP]; pKa = 8.0), would concentrate via proton trapping within lysosomes, and that the vicinal thiols of the reduced form of this agent would interact with intralysosomal iron, preventing oxidant-mediated cell damage. Using a thiol-reactive fluorochrome, we find that reduced LAP does accumulate within the lysosomes of cultured J774 cells. Furthermore, LAP is approximately 1000 and 5000 times more effective than LM and DFO, respectively, in protecting lysosomes against oxidant-induced rupture and in preventing ensuing apoptotic cell death. Suppression of lysosomal accumulation of LAP (by ammonium-mediated lysosomal alkalinization) blocks these protective effects. Electron paramagnetic resonance reveals that the intracellular generation of hydroxyl radical following addition of hydrogen peroxide to J774 cells is totally eliminated by pretreatment with either DFO (1 mM) or LAP (0.2 microM) whereas LM (200 microM) is much less effective.     

Differential cytokine modulation and T cell activation by two distinct classes of thalidomide analogues that are potent inhibitors of TNF-alpha.

TNF-alpha mediates both protective and detrimental manifestations of the host immune response. Our previous work has shown thalidomide to be a relatively selective inhibitor of TNF-alpha production in vivo and in vitro. Additionally, we have recently reported that thalidomide exerts a costimulatory effect on T cell responses. To develop thalidomide analogues with increased anti-TNF-alpha activity and reduced or absent toxicities, novel TNF-alpha inhibitors were designed and synthesized. When a selected group of these compounds was examined for their immunomodulatory activities, different patterns of cytokine modulation were revealed. The tested compounds segregated into two distinct classes: one class of compounds, shown to be potent phosphodiesterase 4 inhibitors, inhibited TNF-alpha production, increased IL-10 production by LPS-induced PBMC, and had little effect on T cell activation; the other class of compounds, similar to thalidomide, were not phosphodiesterase 4 inhibitors and markedly stimulated T cell proliferation and IL-2 and IFN-gamma production. These compounds inhibited TNF-alpha, IL-1beta, and IL-6 and greatly increased IL-10 production by LPS-induced PBMC. Similar to thalidomide, the effect of these agents on IL-12 production was dichotomous; IL-12 was inhibited when PBMC were stimulated with LPS but increased when cells were stimulated by cross-linking the TCR. The latter effect was associated with increased T cell CD40 ligand expression. The distinct immunomodulatory activities of these classes of thalidomide analogues may potentially allow them to be used in the clinic for the treatment of different immunopathological disorders.     

Differential inhibition of mitogen induced T cell proliferation by 5-azacytidine and cytosine-arabinoside

The cytotoxic drugs 5-azacytidine and cytosine-arabinoside influence the enzymatic methylation of DNA in opposite ways (1,2). The in vitro effects of these two drugs on Con A induced proliferation of thymic and splenic rat lymphocytes were investigated. Cytosine-arabinoside was found to inhibit mitogen induced proliferation already at a concentration of 0.001 microM, whereas 5-azacytidine was inhibitory only above concentrations of 1 microM. A stimulation of mitogen induced T cell proliferation was consistently seen with 5-azacytidine, but not with cytosine-arabinoside, at concentrations lower than the cytotoxic concentration. The results show that 5-azacytidine and cytosine-arabinoside interfere with mitogen stimulated lymphocyte proliferation by different mechanisms and suggest that hypomethylated DNA plays a role in the proliferation of T cells.     

Phosphate compounds as iron chelators in animal cell cultures

Summary We have studied the capacity of a number of phosphate compounds to act in the double role as a phosphate source and a detoxifier of ferric chloride hydroxo compounds, i.e. as Fe(III) chelators. The tested compounds were: orthophosphate, trimetaphosphate, α-glycerophosphate, β-glycerophosphate, phytic acid, and phosphorylcholine; the test organism the ciliate protozoonTetrahymena thermophila, an animal cell; and the nutrient medium was synthetic, consisting solely of low-molecular-weight compounds. We assessed growth rates of cells in two experimental series. First, phosphate-starved cells were exposed to the tested phosphate compound as the only phosphate source and the ferric chloride concentrations were varied stepwise from 0 to 1000μM. Second, we offered the cells orthophosphate as a phosphate source and selected phosphate compounds as chelators. The cell growth results allow the following conclusions: orthophosphate, trimetaphosphate, α-glycerophosphate, and β-glycerophosphate are excellent phosphate sources; trimetaphosphate, α-glycerophosphate, β-glycerophosphate, and phytic acid are excellent Fe(III) chelators; of the tested compounds trimetaphosphate, α-glycerophosphate, and β-glycerophosphate are excellent in the double role as a phosphate source and a ferric chloride hydroxo detoxifier, i.e. as a Fe(III) chelator.     

In vitro growth inhibition of bloodstream forms of Trypanosoma brucei and Trypanosoma congolense by iron chelators

African trypanosomes exert significant morbidity and mortality in man and livestock. Only a few drugs are available for the treatment of trypanosome infections and therefore, the development of new anti-trypanosomal agents is required. Previously it has been shown that bloodstream-form trypanosomes are sensitive to the iron chelator deferoxamine. In this study the effect of 13 iron chelators on the growth of Trypanosoma brucei, T. congolense and human HL-60 cells was tested in vitro. With the exception of 2 compounds, all chelators exhibited anti-trypanosomal activities, with 50% inhibitory concentration (IC₅₀) values ranging between 2.1 – 220 μM. However, the iron chelators also displayed cytotoxicity towards human HL-60 cells and therefore, only less favourable selectivity indices compared to commercially available drugs. Interfering with iron metabolism may be a new strategy in the treatment of trypanosome infections. More specifically, lipophilic iron-chelating agents may serve as lead compounds for novel anti-trypanosomal drug development.     

Droplet-based microfluidic platforms for single T cell secretion analysis of IL-10 cytokine

Here we present a microfluidic method for the analysis of single cell secretions. The method co-encapsulates cells with microspheres conjugated with capture antibodies and detection fluorescence-labeled antibodies. The secreted substance captured on the microsphere surface and detected via detection antibodies generating a localized fluorescent signal on a microsphere surface. Using this method, CD4+CD25+ regulatory T cells were encapsulated and assayed to detect IL-10 secreting cell in population.     

Multiple cytokine secretion by IL-7-stimulated human T cells.

The induction of cytokine secretion by human peripheral blood (PB) T cells was examined. Highly purified T cells stimulated with interleukin 7 (IL-7), in the absence of co-mitogen, secreted IL-2, IL-4, IL-6 and interferon gamma (IFN-gamma) upon restimulation with phorbol ester and ionomycin. In contrast, induction of T-cell cultures initiated with IL-2 or IL-4 yielded only low levels of IL-6 and virtually undetectable levels of IL-4 or IFN-gamma, while IL-2 secretion was reduced. No difference was seen in the ability of CD4+ and CD8+ subpopulations, grown in IL-7, to produce cytokines. In contrast, subdivision of T cells into memory and naive populations using the CD45RO monoclonal antibody (mAb) UCHL1, revealed that almost all of the potential to secrete IL-4 and IL-6 in response to IL-7 resided in the CD45RO+ memory population. Stimulation of cytokine-secreting cells appeared to be a direct effect of IL-7 as neutralizing antibodies directed against IL-2 and IL-4 had no effect on the levels of cytokines produced. The differences observed in the ability of IL-2, IL-4, and IL-7 to potentiate cytokine production was supported by measurement of cytokine mRNA levels by PCR. The elevated levels of cytokine secretion seen in cells cultured with IL-7 was not due simply to increased viability in these cultures compared with those containing IL-2 or IL-4, as these populations showed comparable cloning frequencies in phytohemagglutinin (PHA) + IL-2. These results demonstrate that IL-7, in the absence of co-mitogen, is a potent initial stimulus for multiple cytokine production by human T cells upon restimulation.     

SARS- CoV X4 基因转染对T 细胞 TNF- A及IL- 6 分泌的影响

目的：通过检测SARS-CoV X4基因转染后T细胞细胞因子分泌的变化,研究SARS-CoV X4蛋白潜在的功能及其作用机制,并探讨其与SARS-CoV致病机制的关系。方法：利用RosetteSep分离法分离外周血T细胞,以Amaxa核转染仪将pEGFP-SARS-CoV X4真核表达载体转入T细胞,应用激光共聚焦显微镜观察其瞬时表达情况,流式细胞仪检测转染效率,CBA技术检测T细胞细胞因子。结果：SARS-CoV X4基因转染后12h得到了明显表达,共聚焦显微镜观察到EGFP绿色荧光表达,流式细胞仪检测其表达率为245;与空载体转染组相比,SARS-CoV X4基因转染能显著促进T细胞IL-6及TNF-α的分泌。结论：SARS-CoV X4蛋白可诱导T细胞分泌前炎症因子IL-6及TNT-α,SARS-CoV X4蛋白可能在SARS发病机制中起重要的作用。     

Differential sensitivity of T suppressor cell expression to inhibition by histamine type 2 receptor antagonists

The ability of the histamine type 2 (H2) receptor antagonists cimetidine and oxmetidine to inhibit the immune suppression mediated by different types of murine T suppressor cells has been evaluated. Both compounds at doses as low as 1 mg/kg administered as a per os (p.o.) twice a day (b.i.d.) regimen abrogated the expression of dinitrobenzene sulfonic acid-induced, Lyt-2+, T suppressor cells and stimulated contact sensitivity to dinitrofluorobenzene in adoptive transfer experiments. Comparable inhibition of Lyt-1+, T suppressor cell activity induced by UV irradiation required higher doses of cimetidine and oxmetidine (200 and 25 mg/kg; p.o., b.i.d., respectively). In contrast, the T suppressor cell-mediated unresponsiveness induced by inoculation with a high dose of sheep red blood cells was refractory to treatment in vivo with either cimetidine or oxmetidine regardless of the dose. These results indicate that T suppressor cell populations differ markedly in their susceptibility to modulation by H2 antagonists. The histamine type 1 (H1) receptor antagonist diphenhydramine, had no effect on suppressor cell activity in any of these systems, indicating that modulation of suppressor cell activity is mediated through an H2 receptor interaction.     

Regulation of T cell cytokine production by dendritic cells

Previous work has established that the dendritic cells (DC) of mouse spleen regulate the IL-2 production, and hence the extent of proliferation, of the CD8 T cells they activate. It is now reported here that interaction of primary CD8 T cells with splenic CD8alpha- DC induced much higher production of IL-3, IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as IL-2, than did interaction with CD8alpha+ splenic DC. Furthermore, the CD8alpha- DC also induced higher levels of IL-2, IL-3 and IL-10 production in primary CD4 T cells, compared with that induced by CD8alpha+ DC. These quantitative differences did not involve qualitative shifts in the type of cytokine produced. Interleukin-4 production remained low in all the primary T cell cultures and restimulation experiments in secondary cultures did not reveal any bias in the cytokine production profile. When exogenous IL-2 was added to the primary cultures to ensure equal proliferation in response to CD8alpha- or CD8alpha+ DC, the higher level of production of IL-3, IFN-gamma and GM-CSF induced by CD8alpha- DC was maintained. Thus, this general control of T cell cytokine production by splenic DC involves factors additional to those that govern activation of T cells into cell cycle.     

Wortmannin inhibition of forskolin-stimulated chloride secretion by T84 cells

The time- and dose-dependent effects of wortmannin on transepithelial electrical resistance (Rte) and forskolin-stimulated chloride secretion in T84 monolayer cultures were studied. In both instances, maximal effects developed over 2 h and were stable thereafter. Inhibition of forskolin-stimulated chloride secretion, as measured by the short-circuit current (Isc) technique, had an IC50 of 200-500 nM, which is 100-fold higher than for inhibition of phosphatidylinositol 3-kinase (PI3K), but similar to the IC50 for inhibition of myosin light chain kinase (MLCK) and mitogen-activated protein kinases (MAPK). Previous work demonstrated that 500 nM wortmannin did not inhibit the cAMP activation of apical membrane chloride channels. We show here that 500 nM wortmannin has no affect on basolateral Na/K/2Cl-cotransporter activity, but inhibits basolateral membrane Na/K-ATPase activity significantly. The MLCK inhibitors ML-7 and KT5926 were without affect on forskolin-stimulated Isc. Similarly, the p38- and MEK-specific MAPK inhibitors SB203580 and PD98059 did not reduce forskolin-stimulated Isc. In contrast, the non-specific MAPK inhibitor apigenin reduced forskolin-stimulated Isc and basolateral membrane Na/K-ATPase activity similar to wortmannin. In isolated membranes from T84 cells, wortmannin did not inhibit Na/K-ATPase enzymatic activity directly. We conclude that one or more MAPK may regulate the functional expression of basolateral membrane Na/K-ATPase by controlling the abundance of enzyme molecules in the plasma membrane.     

ROCKing cytokine secretion balance in human T cells

Balanced regulation of cytokine secretion in T cells is critical for maintenance of immune homeostasis and prevention of autoimmunity. The Rho-associated kinase (ROCK) 2 signaling pathway was previously shown to be involved in controlling of cellular movement and shape. However, recent work from our group and others has demonstrated a new and important role of ROCK2 in regulating cytokine secretion in T cells. We found that ROCK2 promotes pro-inflammatory cytokines such as IL-17 and IL-21, whereas IL-2 and IL-10 secretion are negatively regulated by ROCK2 under Th17-skewing activation. Also, in disease, but not in steady state conditions, ROCK2 contributes to regulation of IFN-γ secretion in T cells from rheumatoid arthritis patients. Thus, ROCK2 signaling is a key pathway in modulation of T-cell mediated immune responses underscoring the therapeutic potential of targeted inhibition of ROCK2 in autoimmunity.     

Differential inhibition of human antigen-specific T cell clone proliferative responses by distinct monoclonal anti-HLA-DR antibodies

The inhibition of diphtheria toxoid and varidase-specific T cell clones from a single DR 6/7 donor by eight distinct monoclonal anti-HLA-DR antibodies was tested in proliferative assays. These moAb were selected because they had been previously defined for their ability: 1) to react with Ia molecules; 2) to recognize similar or different epitopes; 3) to share or not share idiotypic specificities. Our results show a distinct inhibition pattern for each clone tested. Furthermore, the various moAb could be classified into three groups according to their inhibitory effect on T cell proliferation. These data suggest: a) an epitopic restriction by class II antigens of antigen-specific human T cell clone proliferation; and b) the recognition of functional epitopes on the human Ia-like antigens by some but not all moAb studied.