VDAC1-interacting molecules promote cell death in cancer organoids through mitochondrial-dependent metabolic interference.

The voltage-dependent anion-selective channel isoform 1 (VDAC1) is a pivotal component in cellular metabolism and apoptosis with a prominent role in many cancer types, offering a unique therapeutic intervention point. Through an in-silico-to-in-vitro approach we identified a set of VA molecules (VDAC Antagonists) that selectively bind to VDAC1 and display specificity toward cancer cells. Biochemical characterization showed that VA molecules can directly interact with VDAC1 with micromolar affinity by competing with the endogenous ligand NADH for a partially shared binding site. NADH displacement results in mitochondrial distress and reduced cell proliferation, especially when compared to non-cancerous cells. Experiments performed on organoids derived from intrahepatic cholangiocarcinoma patients demonstrated a dose-dependent reduction in cell viability upon treatment with VA molecules with lower impact on healthy cells than conventional treatments like gemcitabine. VA molecules are chemical entities representing promising candidates for further optimization and development as cancer therapy strategies through precise metabolic interventions.

The protease activated receptor 2 - CCAAT/enhancer-binding protein beta - SerpinB3 axis inhibition as a novel strategy for the treatment of non-alcoholic steatohepatitis.

OBJECTIVE: The serine protease inhibitor SerpinB3 has been described as critical mediator of liver fibrosis and it has been recently proposed as an additional hepatokine involved in NASH development and insulin resistance. Protease Activated Receptor 2 has been identified as a novel regulator of hepatic metabolism. A targeted therapeutic strategy for NASH has been investigated, using 1-Piperidine Propionic Acid (1-PPA), since this compound has been recently proposed as both Protease Activated Receptor 2 and SerpinB3 inhibitor. METHODS: The effect of SerpinB3 on inflammation and fibrosis genes was assessed in human macrophage and stellate cell lines. Transgenic mice, either overexpressing SerpinB3 or carrying Serpinb3 deletion and their relative wild type strains, were used in experimental NASH models. Subgroups of SerpinB3 transgenic mice and their controls were also injected with 1-PPA to assess the efficacy of this compound in NASH inhibition. RESULTS: 1-PPA did not present significant cell and organ toxicity and was able to inhibit SerpinB3 and PAR2 in a dose-dependent manner. This effect was associated to a parallel reduction of the synthesis of the molecules induced by endogenous SerpinB3 or by its paracrine effects both in vitro and in vivo, leading to inhibition of lipid accumulation, inflammation and fibrosis in experimental NASH. At mechanistic level, the antiprotease activity of SerpinB3 was found essential for PAR2 activation, determining upregulation of the CCAAT Enhancer Binding Protein beta (C/EBP-ß), another pivotal regulator of metabolism, inflammation and fibrosis, which in turn determined SerpinB3 synthesis. CONCLUSIONS: 1-PPA treatment was able to inhibit the PAR2 - C/EBP-ß - SerpinB3 axis and to protect from NASH development and progression, supporting the potential use of a similar approach for a targeted therapy of NASH.

Chronic Liver Disease: Latest Research in Pathogenesis, Detection and Treatment.

Chronic liver disease (CLD) is a major global health threat and has emerged as a leading cause of human death [...].

Liver Organoids as an In Vitro Model to Study Primary Liver Cancer.

Primary liver cancers (PLC), including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), are among the leading causes of cancer-related mortality worldwide. Bi-dimensional in vitro models are unable to recapitulate the key features of PLC; consequently, recent advancements in three-dimensional in vitro systems, such as organoids, opened up new avenues for the development of innovative models for studying tumour's pathological mechanisms. Liver organoids show self-assembly and self-renewal capabilities, retaining essential aspects of their respective in vivo tissue and allowing modelling diseases and personalized treatment development. In this review, we will discuss the current advances in the field of liver organoids focusing on existing development protocols and possible applications in regenerative medicine and drug discovery.

Characterization of Mitochondrial Alterations in Aicardi-Goutières Patients Mutated in RNASEH2A and RNASEH2B Genes.

Aicardi-Goutières syndrome (AGS) is a rare encephalopathy characterized by neurological and immunological features. Mitochondrial dysfunctions may lead to mitochondrial DNA (mtDNA) release and consequent immune system activation. We investigated the role of mitochondria and mtDNA in AGS pathogenesis by studying patients mutated in RNASEH2B and RNASEH2A genes. Lymphoblastoid cell lines (LCLs) from RNASEH2A- and RNASEH2B-mutated patients and healthy control were used. Transmission Electron Microscopy (TEM) and flow cytometry were used to assess morphological alterations, reactive oxygen species (ROS) production and mitochondrial membrane potential variations. Seahorse Analyzer was used to investigate metabolic alterations, and mtDNA oxidation and VDAC1 oligomerization were assessed by immunofluorescence. Western blot and RT-qPCR were used to quantify mtTFA protein and mtDNA release. Morphological alterations of mitochondria were observed in both mutated LCLs, and loss of physiological membrane potential was mainly identified in RNASEH2A LCLs. ROS production and 8-oxoGuanine levels were increased in RNASEH2B LCLs. Additionally, the VDAC1 signal was increased, suggesting a mitochondrial pore formation possibly determining mtDNA release. Indeed, higher cytoplasmic mtDNA levels were found in RNASEH2B LCLs. Metabolic alterations confirmed mitochondrial damage in both LCLs. Data highlighted mitochondrial alterations in AGS patients' LCLs suggesting a pivotal role in AGS pathogenesis.

SerpinB3 administration protects liver against ischemia-reperfusion injury.

We have investigated the change in SerpinB3 during hepatic ischemia and the potential role of its antiprotease activity in cell protection by the administration of wild-type SerpinB3 (SerpinB3-WT) or active loop-deleted recombinant SerpinB3 protein (SerpinB3-D) in a rat model of ischemia (60 min)/reperfusion (60 min) (I/R). A time-dependent increase of SerpinB3, both at transcription and protein level, was found in ischemic livers after 60, 120 and 180 min. SerpinB3-WT decreased polymorphonuclear cell infiltration and serum enzymes and increased ATP when compared with I/R group. These events were not obtained using SerpinB3-D. No significant changes in both liver SerpinB3 mRNA and protein were found in all I/R groups considered. The present data show that the administration of SerpinB3-WT reduced the I/R injury and this effect appears to be dependent on its anti-protease activity.