Ligand-Receptor Interactions of Galectin-9 and VISTA Suppress Human T Lymphocyte Cytotoxic Activity.

Acute myeloid leukemia (AML), a blood/bone marrow cancer, is a severe and often fatal malignancy. AML cells are capable of impairing the anti-cancer activities of cytotoxic lymphoid cells. This includes the inactivation of natural killer (NK) cells and killing of T lymphocytes. Here we report for the first time that V-domain Ig-containing suppressor of T cell activation (VISTA), a protein expressed by T cells, recognizes galectin-9 secreted by AML cells as a ligand. Importantly, we found that soluble VISTA released by AML cells enhances the effect of galectin-9, most likely by forming multiprotein complexes on the surface of T cells and possibly creating a molecular barrier. These events cause changes in the plasma membrane potential of T cells leading to activation of granzyme B inside cytotoxic T cells, resulting in apoptosis.

Synthesis and in Vitro Bioactivity of Polyunsaturated Fatty Acid Conjugates of Combretastatin A-4.

Combretastatin A-4 (CA-4) (1) is a plant-derived anticancer agent binding to the tubulin colchicine site. Polyunsaturated fatty acids (PUFAs) are readily taken up by cancer cells and have been used to improve cell targeting. In the present study, four CA-4-PUFA conjugates were synthesized by coupling combretastatin A-4 (1) with several polyunsaturated fatty acids. The conjugates (2a-d) were characterized using spectroscopic methods. Their cytotoxicity was evaluated against human breast cancer cells (MCF-7), and the inhibition of tubulin polymerization was determined in vitro. All conjugates influenced tubulin polymerization, with the arachidonic acid conjugate (2c) displaying cytotoxicity similar in potency to the natural product CA-4 (1).

Caffeine affects the biological responses of human hematopoietic cells of myeloid lineage via downregulation of the mTOR pathway and xanthine oxidase activity.

Correction of human myeloid cell function is crucial for the prevention of inflammatory and allergic reactions as well as leukaemia progression. Caffeine, a naturally occurring food component, is known to display anti-inflammatory effects which have previously been ascribed largely to its inhibitory actions on phosphodiesterase. However, more recent studies suggest an additional role in affecting the activity of the mammalian target of rapamycin (mTOR), a master regulator of myeloid cell translational pathways, although detailed molecular events underlying its mode of action have not been elucidated. Here, we report the cellular uptake of caffeine, without metabolisation, by healthy and malignant hematopoietic myeloid cells including monocytes, basophils and primary acute myeloid leukaemia mononuclear blasts. Unmodified caffeine downregulated mTOR signalling, which affected glycolysis and the release of pro-inflammatory/pro-angiogenic cytokines as well as other inflammatory mediators. In monocytes, the effects of caffeine were potentiated by its ability to inhibit xanthine oxidase, an enzyme which plays a central role in human purine catabolism by generating uric acid. In basophils, caffeine also increased intracellular cyclic adenosine monophosphate (cAMP) levels which further enhanced its inhibitory action on mTOR. These results demonstrate an important mode of pharmacological action of caffeine with potentially wide-ranging therapeutic impact for treating non-infectious disorders of the human immune system, where it could be applied directly to inflammatory cells.

Development of poly(glycerol adipate) nanoparticles loaded with non-steroidal anti-inflammatory drugs.

The aim of this study was to assess acylated and non-acylated poly(glycerol adipate) polymers (PGA) as suitable nanoparticulate systems for encapsulation and release of ibuprofen, ibuprofen sodium salt (IBU-Na) and ketoprofen as model drugs. Drug encapsulated nanoparticles were prepared using the interfacial deposition method in the absence of surfactants. Physicochemical characterisation studies of the produced loaded nanoparticles showed that drug-polymer interactions depend on the characteristics of the actual active substance. IBU-Na showed strong interactions with the polymers and it was found to be molecularly dispersed within the polymer matrix while ibuprofen and ketoprofen retained their crystalline state. The drug release profiles showed stepwise patterns which involve an initial burst release effect, diffusion of the drug from the polymer matrix and eventually drug release possibly via a combined mechanism. PGA polymers can be effectively used as drug delivery carriers for various active substances.

Azinomycin epoxide induces activation of apoptosis signal-regulating kinase 1 (ASK1) and caspase 3 in a HIF-1alpha-independent manner in human leukaemia myeloid macrophages.

In recent years there has been an increasing interest in the azinomycin epoxide (2S, 3S)-benzyl 3,4-epoxy-2-(3-methoxy-5-methyl-1-naphthoyloxy)-3-methylbut anamide (EA), a potent cytotoxic and anti-tumour antibiotic. However, the molecular mechanisms of its cytotoxic activity have not yet been investigated. Here we report that exposure of the THP-1 human myeloid leukaemia cells to EA leads to the activation of apoptosis signal-regulating kinase 1 via a redox-dependent mechanism in a concentration and time-dependent manner. Accumulation of p53 and activation of caspase 3 were also seen. This was consistent with EA concentration-dependent accumulation of HIF-1alpha protein peaking after 4 h of stimulation with EA. Experiments with THP-1 cells, where the activity of ASK1 was blocked by transfection with the dominant-negative form of ASK1 demonstrated the importance of this enzyme for EA-dependent activation of caspase 3. Accumulated HIF-1alpha protein did not, however, promote EA-induced activation of caspase 3 or accumulation of p53. The experiments with p53 knockdown THP-1 cells demonstrated that this protein is not important for EA-induced activation of ASK1, caspase 3 or accumulation of HIF-1alpha protein. This is consistent with previous results indicating a reduced activity of p53 in THP-1 cells.

Design and synthesis of a DNA-crosslinking azinomycin analogue.

The azinomycins are potent antitumour antibiotics that are able to crosslink DNA, but are relatively unstable and unlikely to progress as therapeutic candidates. A prototype analogue 4 with more clinical potential has been designed and synthesised and incorporates the epoxide function of the azinomycins and a nitrogen mustard. Two further analogues 5 and 6 that can alkylate DNA but cannot crosslink the duplex have also been synthesised. Compound 4 crosslinks DNA efficiently at nM concentrations. Compounds 4-6 were submitted to the NCI 60 cell line screen and have similar antitumour activity, although 4 is slightly less active than the non-crosslinking compounds. These observations will be important in the design of further azinomycin analogues with antitumour activity.

Truncated azinomycin analogues intercalate into DNA.

The design and synthesis of a potentially more therapeutically-viable azinomycin analogue 4 based upon 3 has been completed. It involved coupling of a piperidine mustard to the acid chloride of the azinomycin chromophore. Both the designed azinomycin analogue 4 and the natural product 3 bind to DNA and cause unwinding, supporting an intercalative mode of binding.