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Tumour-associated macrophages mainly comprise immunosuppressive M2 phenotypes that promote tumour progression besides anti-tumoural M1 subsets. Selective depletion or reprogramming of M2 may represent an innovative anti-cancer strategy. The actin cytoskeleton is central for cellular homeostasis and is targeted for anti-cancer chemotherapy. Here, we show that targeting G-actin nucleation using chondramide A (ChA) predominantly depletes human M2 while promoting the tumour-suppressive M1 phenotype. ChA reduced the viability of M2, with minor effects on M1, but increased tumour necrosis factor (TNF)α release from M1. Interestingly, ChA caused rapid disruption of dynamic F-actin filaments and polymerization of G-actin, followed by reduction of cell size, binucleation and cell division, without cellular collapse. In M1, but not in M2, ChA caused marked activation of SAPK/JNK and NFκB, with slight or no effects on Akt, STAT-1/-3, ERK-1/2, and p38 MAPK, seemingly accounting for the better survival of M1 and TNFα secretion. In a microfluidically-supported human tumour biochip model, circulating ChA-treated M1 markedly reduced tumour cell viability through enhanced release of TNFα. Together, ChA may cause an anti-tumoural microenvironment by depletion of M2 and activation of M1, suggesting induction of G-actin nucleation as potential strategy to target tumour-associated macrophages in addition to neoplastic cells., Competing Interests: The authors declare no competing financial interests.

Full details on the evaluation and application of an easily feasible and generally useful method for configurational assignments of isolated methyl-bearing stereocenters are reported. The analytical tool relies on a bioinformatic gene cluster analysis and utilizes a predictive enoylreductase alignment, and its feasibility was demonstrated by the full stereochemical determination of the ajudazols, highly potent inhibitors of the mitochondrial respiratory chain. Furthermore, a full account of our strategies and tactics that culminated in the total synthesis of ajudazol B, the most potent and least abundant of these structurally unique class of myxobacterial natural products, is presented. Key features include an application of an asymmetric ortholithiation strategy for synthesis of the characteristic anti-configured hydroxyisochromanone core bearing three contiguous stereocenters, a modular oxazole formation, a flexible cross-metathesis approach for terminal allyl amide synthesis, and a late-stage Z,Z-selective Suzuki coupling. This total synthesis unambiguously proves the correct stereochemistry, which was further corroborated by comparison with reisolated natural material. Finally, 5-lipoxygenase was discovered as an additional molecular target of ajudazol B. Activities against this clinically validated key enzyme of the biosynthesis of proinflammatory leukotrienes were in the range of the approved drug zileuton, which further underlines the biological importance of this unique natural product.

Monocytes are an important constituent of the innate immune system. Therefore, manipulating gene expression of primary human monocytes is a crucial mean to study and characterize the functions of targeted proteins in monocytes. Gene silencing by transfection of cells with small interfering RNA (siRNA) leading to the degradation of the corresponding mRNA and thus to reduced target protein levels is an important tool to investigate gene and protein function of interest. However, non-viral transfection of primary monocytes is challenging because siRNA uptake by these suspended cells is tricky, and the individual cells vary among different donors and do not proliferate. Here, we describe a procedure for efficient non-viral transfection of primary human monocytes isolated from peripheral blood, which maintains cell viability and cell functions, such as responsiveness to stimuli like LPS and IL-10. Nucleofection was used as an electroporation technique that enables efficient introduction of siRNA and silencing of target genes. Using a modification of our previously published protocol for the fast-proliferating THP-1 monocytic cell line, we transfected primary human monocytes with siRNA targeting 5-lipoxygenase (5-LO). In fact, we successfully downregulated 5-LO mRNA resulting in reduced protein levels and enzymatic activity., (Copyright © 2015 Elsevier B.V. All rights reserved.)