Induction of apoptosis in murine leukemia by diarylheptanoids from Curcuma comosa Roxb.

Diarylheptanoids, isolated from the rhizome of Curcuma comosa Roxb., have several biological activities including anti-oxidant and anti-inflammation. The present study investigated the effect of five diarylheptanoids isolated from C. comosa rhizome on the proliferation of murine P388 leukemic cells. Compound-092, (3S)-1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol, bearing a catechol moiety, was the most potent diarylheptanoid (IC₅₀ of 4 μM) in inhibiting P388 leukemic cell viability by causing DNA breakage and inducing apoptosis. Apoptotic cell death was characterized by the presence of chromatin condensation, formation of apoptotic bodies, DNA fragmentation, and externalization of plasma membrane phosphatidylserine. This compound increased caspase-3 activity about fivefold above the untreated control, decreased the intracellular reduced glutathione level, and impaired mitochondrial transmembrane potential. In the presence of Cu(II) ion, the compound exhibited a pro-oxidant activity causing DNA strand breakage and enhancing the anti-proliferative activity. The results provide evidence for the pro-oxidant activity of the diarylheptanoid bearing a catechol moiety in the induction of apoptosis in murine P388 leukemia.     

Anoctamin-6 Controls Bone Mineralization by Activating the Calcium Transporter NCX1

Anoctamin-6 (Ano6, TMEM16F) belongs to a family of putative Ca²⁺-activated Cl⁻ channels and operates as membrane phospholipid scramblase. Deletion of Ano6 leads to reduced skeleton size, skeletal deformities, and mineralization defects in mice. However, it remains entirely unclear how a lack of Ano6 leads to a delay in bone mineralization by osteoblasts. The Na⁺/Ca²⁺ exchanger NCX1 was found to interact with Ano6 in a two-hybrid split-ubiquitin screen. Using human osteoblasts and osteoblasts from Ano6^−/− and WT mice, we demonstrate that NCX1 requires Ano6 to efficiently translocate Ca²⁺ out of osteoblasts into the calcifying bone matrix. Ca²⁺-activated anion currents are missing in primary osteoblasts isolated from Ano6 null mice. Our findings demonstrate the importance of NCX1 for bone mineralization and explain why deletion of an ion channel leads to the observed mineralization defect: Ano6 Cl⁻ currents are probably required to operate as a Cl⁻ bypass channel, thereby compensating net Na⁺ charge movement by NCX1.     

Role of anoctamins in cancer and apoptosis

Anoctamin 1 (TMEM16A, Ano1) is a recently identified Ca²⁺-activated chloride channel and a member of a large protein family comprising 10 paralogues. Before Ano1 was identified as a chloride channel protein, it was known as the cancer marker DOG1. DOG1/Ano1 is expressed in gastrointestinal stromal tumours (GIST) and particularly in head and neck squamous cell carcinoma, at very high levels never detected in other tissues. It is now emerging that Ano1 is part of the 11q13 locus, amplified in several types of tumour, where it is thought to augment cell proliferation, cell migration and metastasis. Notably, Ano1 is upregulated through histone deacetylase (HDAC), corresponding to the known role of HDAC in HNSCC. As Ano1 does not enhance proliferation in every cell type, its function is perhaps modulated by cell-specific factors, or by the abundance of other anoctamins. Thus Ano6, by regulating Ca²⁺-induced membrane phospholipid scrambling and annexin V binding, supports cellular apoptosis rather than proliferation. Current findings implicate other cellular functions of anoctamins, apart from their role as Ca²⁺-activated Cl⁻ channels.     

A novel microscopy-based assay identifies extended synaptotagmin-1 (ESYT1) as a positive regulator of anoctamin 1 traffic

An attractive possibility to treat Cystic Fibrosis (CF), a severe condition caused by dysfunctional CFTR, an epithelial anion channel, is through the activation of alternative (non-CFTR) anion channels. Anoctamin 1 (ANO1) was demonstrated to be a Ca²⁺-activated chloride channel (CaCC) and thus of high potential to replace CFTR. Despite that ANO1 is expressed in human lung CF tissue, it is present at the cell surface at very low levels. In addition, little is known about regulation of ANO1 traffic, namely which factors promote its plasma membrane (PM) localization.Here, we generated a novel cellular model, expressing an inducible 3HA-ANO1-eGFP construct, and validated its usage as a microscopy tool to monitor for ANO1 traffic.We demonstrate the robustness and specificity of this cell-based assay, by the identification of siRNAs acting both as ANO1 traffic enhancer and inhibitor, targeting respectively COPB1 and ESYT1 (extended synaptotagmin-1), the latter involved in coupling of the endoplasmic reticulum to the PM at specific microdomains. We further show that knockdown of ESYT1 (and family members ESYT2 and ESYT3) significantly decreased ANO1 current density.This ANO1 cell-based assay constitutes an important tool to be further used in high-throughput screens and drug discovery of high relevance for CF and cancer.     

A Coding Variant of ANO10, Affecting Volume Regulation of Macrophages,Is Associated with Borrelia Seropositivity

In a first genome-wide association study (GWAS) approach to anti-Borrelia seropositivity, we identified two significant single nucleotide polymorphisms (SNPs) (rs17850869, P = 4.17E-09; rs41289586, P = 7.18E-08). Both markers, located on chromosomes 16 and 3, respectively, are within or close to genes previously connected to spinocerebellar ataxia. The risk SNP rs41289586 represents a missense variant (R263H) of anoctamin 10 (ANO10), a member of a protein family encoding Cl⁻ channels and phospholipid scram-blases. ANO10 augments volume-regulated Cl⁻ currents (I_Hypo) in Xenopus oocytes, HEK293 cells, lymphocytes and macrophages and controls volume regulation by enhancing regulatory volume decrease (RVD). ANO10 supports migration of macrophages and phagocytosis of spirochetes. The R263H variant is inhibitory on I_Hypo, RVD and intracellular Ca²⁺ signals, which may delay spirochete clearance, thereby sensitizing adaptive immunity. Our data demonstrate for the first time that ANO10 has a central role in innate immune defense against Borrelia infection.