Protein misfolding diseases, including α1-antitrypsin deficiency (AATD), pose substantial health challenges, with their cellular progression still poorly understood1-3. We use spatial proteomics by mass spectrometry and machine learning to map AATD in human liver tissue. Combining Deep Visual Proteomics (DVP) with single-cell analysis4,5, we probe intact patient biopsies to resolve molecular events during hepatocyte stress in pseudotime across fibrosis stages. We achieve proteome depth of up to 4,300 proteins from one-third of a single cell in formalin-fixed, paraffin-embedded tissue. This dataset reveals a potentially clinically actionable peroxisomal upregulation that precedes the canonical unfolded protein response. Our single-cell proteomics data show α1-antitrypsin accumulation is largely cell-intrinsic, with minimal stress propagation between hepatocytes. We integrated proteomic data with artificial intelligence-guided image-based phenotyping across several disease stages, revealing a late-stage hepatocyte phenotype characterized by globular protein aggregates and distinct proteomic signatures, notably including elevated TNFSF10 (also known as TRAIL) amounts. This phenotype may represent a critical disease progression stage. Our study offers new insights into AATD pathogenesis and introduces a powerful methodology for high-resolution, in situ proteomic analysis of complex tissues. This approach holds potential to unravel molecular mechanisms in various protein misfolding disorders, setting a new standard for understanding disease progression at the single-cell level in human tissue.

• MeSH
• alpha 1-Antitrypsin metabolism   MeSH
• Single-Cell Analysis MeSH
• alpha 1-Antitrypsin Deficiency * pathology   metabolism   genetics   MeSH
• Phenotype MeSH
• Hepatocytes metabolism   pathology   MeSH
• Liver Cirrhosis pathology   metabolism   MeSH
• Liver pathology   metabolism   MeSH
• Humans MeSH
• Disease Progression MeSH
• Proteome * analysis   metabolism   MeSH
• Proteomics * methods   MeSH
• Unfolded Protein Response MeSH
• Machine Learning MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Infectious diseases, including bacterial, fungal, and viral, have once again gained urgency in the drug development pipeline after the recent COVID-19 pandemic. Tuberculosis (TB) is an old infectious disease for which eradication has not yet been successful. Novel agents are required to have potential activity against both drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of TB. In this study, we present a series of 2-phenyl-N-(pyridin-2-yl)acetamides in an attempt to investigate their possible antimycobacterial activity, cytotoxicity on the HepG2 liver cancer cell line, and-as complementary testing-their antibacterial and antifungal properties against a panel of clinically important pathogens. This screening resulted in one compound with promising antimycobacterial activity-compound 12, MICMtb H37Ra = 15.625 μg/mL (56.26 μM). Compounds 17, 24, and 26 were further screened for their antiproliferative activity against human epithelial kidney cancer cell line A498, human prostate cancer cell line PC-3, and human glioblastoma cell line U-87MG, where they were found to possess interesting activity worth further exploration in the future.

• MeSH
• Acetamides * chemistry   pharmacology   MeSH
• Antifungal Agents pharmacology   chemistry   chemical synthesis   MeSH
• Antitubercular Agents pharmacology   chemistry   MeSH
• Hep G2 Cells MeSH
• Humans MeSH
• Microbial Sensitivity Tests * MeSH
• Mycobacterium tuberculosis * drug effects   MeSH
• Cell Line, Tumor MeSH
• Cell Proliferation * drug effects   MeSH
• Antineoplastic Agents pharmacology   chemistry   MeSH
• Pyridines chemistry   pharmacology   MeSH
• SARS-CoV-2 drug effects   MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

LIM and Src homology 3 (SH3) protein 2 (LASP2) is a small focal adhesion protein first identified as a splice variant of the nebulette gene (Nebl). As the newest member of the nebulin protein family, the regulation and function of LASP2 remain largely unknown. Our previous RNA-sequencing results identified Nebl as one of the most highly induced genes in the mouse liver in response to the activation of pregnane X receptor (PXR). In this study, we investigated this phenomenon further and show that PXR induces Lasp2 instead of Nebl, which partially use the same exons. Lasp2 was found to be induced in response to PXR ligand pregnenolone 16α-carbonitrile (PCN) treatment in mouse liver in vivo both after 4-day treatment and after long-term, 28-day treatment and in both male and female mice. Interestingly, the Lasp2 induction was more efficient in high-fat diet-fed mice (103-fold after 4-day PCN treatment) than in the normal chow-fed mice (32-fold after 4-day PCN treatment). Lasp2 induction was abolished in PXR knockout mice but could be rescued by re-expression of PXR, indicating that Lasp2 induction is PXR mediated. In mouse primary hepatocytes cycloheximide did not inhibit Lasp2 induction by PCN and a PXR binding site could be recognized upstream of the mouse Lasp2 gene suggesting direct regulation of Lasp2 by PXR. In human 3D hepatocytes, rifampicin induced only a modest increase in LASP2 expression. This study shows for the first time that PXR activation strongly induces Lasp2 expression in mouse liver and establishes Lasp2 as a novel PXR target gene. SIGNIFICANCE STATEMENT: RNA-sequencing results have previously identified nebulette (Nebl) to be efficiently induced by pregnane X receptor activating compounds. This study shows that instead of Nebl, LIM and Src homology 3 (SH3) protein 2 (Lasp2) coding for a small focal adhesion protein and partly sharing exons with the Nebl gene is a novel target of pregnane X receptor in mouse liver.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• Cytoskeletal Proteins * genetics   metabolism   MeSH
• Hepatocytes metabolism   drug effects   MeSH
• Liver * metabolism   drug effects   MeSH
• Humans MeSH
• Mice, Inbred C57BL * MeSH
• Mice, Knockout * MeSH
• Mice MeSH
• Pregnane X Receptor * genetics   metabolism   MeSH
• Pregnenolone Carbonitrile pharmacology   MeSH
• LIM Domain Proteins * genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Hepatocellular carcinoma (HCC) cells critically depend on PARP1 and CHK1 activation for survival. Combining the PARP inhibitor (PARPi) olaparib with a CHK1 inhibitor (MK-8776, CHK1i) produced a synergistic effect, reducing cell viability and inducing marked oxidative stress and DNA damage, particularly in the HepG2 cells. This dual treatment significantly increased apoptosis markers, including γH2AX and caspase-3/7 activity. Both HCC cell lines exhibited heightened sensitivity to the combined treatment. The effect of drugs on the expression of proliferation markers in an olaparib-resistant patient-derived xenograft (PDX) model of ovarian cancer was also investigated. Ovarian tumors displayed reduced tissue growth, as reflected by a drop in proliferation marker Ki-67 levels in response to PARPi combined with CHK1i. No changes were observed in corresponding liver tissues using Ki-67 and pCHK staining, which indicates the absence of metastases and a hepatotoxic effect. Thus, our results indicate that the dual inhibition of PARP and CHK1 may prove to be a promising therapeutic approach in the treatment of primary HCC as well as OC tumors without the risk of liver metastases, especially in patients with olaparib-resistant tumor profiles.

• MeSH
• Apoptosis drug effects   MeSH
• Hep G2 Cells MeSH
• Checkpoint Kinase 1 metabolism   antagonists & inhibitors   MeSH
• Phthalazines * pharmacology   MeSH
• Carcinoma, Hepatocellular * drug therapy   pathology   metabolism   MeSH
• Liver drug effects   pathology   metabolism   MeSH
• Humans MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Liver Neoplasms * drug therapy   pathology   metabolism   MeSH
• Ovarian Neoplasms * drug therapy   pathology   metabolism   MeSH
• Poly(ADP-ribose) Polymerase Inhibitors pharmacology   therapeutic use   MeSH
• Piperazines * pharmacology   MeSH
• DNA Damage drug effects   MeSH
• Cell Proliferation drug effects   MeSH
• Antineoplastic Combined Chemotherapy Protocols pharmacology   therapeutic use   adverse effects   MeSH
• Pyrazoles pharmacology   MeSH
• Pyrimidines MeSH
• Drug Synergism * MeSH
• Cell Survival drug effects   MeSH
• Xenograft Model Antitumor Assays * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Hepatitis B virus (HBV) infection can cause liver disease and lead to hepatocellular carcinoma (HCC). To better understand the factors involved in viral infection and pathogenesis and to develop novel therapies, it is crucial to investigate virus-host interactions. HBV infection has been shown to increase the expression of the unconventional prefoldin RPB5 interactor (URI1), a cellular protein that promotes liver tumorigenesis and HCC metastasis. Our study investigated the role of URI1 in HBV infection in vitro. Although previous reports have suggested that URI1 may act as an HBV restriction factor, our results showed that URI1 silencing or overexpression did not affect HBV replication in HepG2-NTCP cells. In primary human hepatocytes, URI1 knockdown modestly reduced HBV markers but did not significantly alter acute infection. Supporting the premise that URI1 is a promising therapeutic target for HCC, our findings show that URI1 knockdown does not enhance HBV infection in an acute infection model. This suggests that URI1 may be a viable therapeutic target for patients with HBV-associated HCC without increasing HBV-related complications.

• MeSH
• Hep G2 Cells MeSH
• Gene Knockdown Techniques MeSH
• Hepatitis B * virology   complications   metabolism   MeSH
• Carcinoma, Hepatocellular virology   metabolism   MeSH
• Hepatocytes * virology   metabolism   MeSH
• Host-Pathogen Interactions MeSH
• Humans MeSH
• Virus Replication * MeSH
• Hepatitis B virus * genetics   physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Endocrine-disrupting chemicals (EDCs) may contribute to the rising incidence of metabolic dysfunction-associated steatotic liver disease (MASLD). We investigated the potential of 10 environmentally relevant EDCs to affect key events of hepatic steatosis in HepG2 human hepatoblastoma cells. Increased lipid droplet formation, a key marker of steatosis, was induced by PFOA, bisphenol F, DDE, butylparaben, and DEHP, within the non-cytotoxic concentration range of 1 nM-25 μM. Cadmium also induced this effect, but at concentrations impairing cell viability (>1 μM). At non-cytotoxic concentrations, these compounds, along with bisphenol A, dysregulated major genes controlling lipid homeostasis. Cadmium, PFOA, DDE, and DEHP significantly upregulated the DGAT1 gene involved in triglyceride synthesis, while butylparaben increased the expression of the FAT/CD36 gene responsible for fatty acid uptake. Bisphenol A downregulated the CPT1A gene involved in fatty acid oxidation. No significant effects on lipid droplet accumulation or lipid metabolism-related genes were observed for PFOS, bisphenol S, and dibutyl phthalate. Among the tested EDCs, lipid accumulation positively correlated with the expression of SREBF1, DGAT1, and CPT1A. These findings provide additional evidence that EDCs can affect MASLD and highlight the utility of in vitro methods in the screening of EDCs with hazardous steatogenic and metabolism-disrupting properties.

• MeSH
• Benzhydryl Compounds toxicity   MeSH
• Hep G2 Cells MeSH
• Diacylglycerol O-Acyltransferase genetics   metabolism   MeSH
• Diethylhexyl Phthalate toxicity   MeSH
• Endocrine Disruptors * toxicity   MeSH
• Phenols toxicity   MeSH
• Fluorocarbons toxicity   MeSH
• Caprylates toxicity   MeSH
• Humans MeSH
• Lipid Metabolism drug effects   MeSH
• Bisphenol A Compounds MeSH
• Cell Survival drug effects   MeSH
• Fatty Liver * chemically induced   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Fibrosis contributes to tissue repair, but excessive fibrosis disrupts organ function. Alagille syndrome (ALGS, caused by mutations in JAGGED1) results in liver disease and characteristic fibrosis. Here, we show that Jag1Ndr/Ndr mice, a model for ALGS, recapitulate ALGS-like fibrosis. Single-cell RNA-seq and multi-color flow cytometry of the liver revealed immature hepatocytes and paradoxically low intrahepatic T cell infiltration despite cholestasis in Jag1Ndr/Ndr mice. Thymic and splenic regulatory T cells (Tregs) were enriched and Jag1Ndr/Ndr lymphocyte immune and fibrotic capacity was tested with adoptive transfer into Rag1-/- mice, challenged with dextran sulfate sodium (DSS) or bile duct ligation (BDL). Transplanted Jag1Ndr/Ndr lymphocytes were less inflammatory with fewer activated T cells than Jag1+/+ lymphocytes in response to DSS. Cholestasis induced by BDL in Rag1-/- mice with Jag1Ndr/Ndr lymphocytes resulted in periportal Treg accumulation and three-fold less periportal fibrosis than in Rag1-/- mice with Jag1+/+ lymphocytes. Finally, the Jag1Ndr/Ndr hepatocyte expression profile and Treg overrepresentation were corroborated in patients' liver samples. Jag1-dependent hepatic and immune defects thus interact to determine the fibrotic process in ALGS.

• MeSH
• Alagille Syndrome pathology   genetics   MeSH
• Cell Differentiation * MeSH
• Hepatocytes * metabolism   pathology   MeSH
• Liver Cirrhosis * pathology   genetics   MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Jagged-1 Protein * metabolism   genetics   MeSH
• T-Lymphocytes, Regulatory immunology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Various strategies have been employed to improve the reliability of 2D, 3D, and co-culture in vitro models of nonalcoholic fatty liver disease, including using extracellular matrix proteins such as collagen I to promote cell adhesion. While studies have demonstrated the significant benefits of culturing cells on collagen I, its effects on the HepG2 cell line after exposure to palmitate (PA) have not been investigated. Therefore, this study aimed to assess the effects of PA-induced lipotoxicity in HepG2 cultured in the absence or presence of collagen I. HepG2 cultured in the absence or presence of collagen I was exposed to PA, followed by analyses that assessed cell proliferation, viability, adhesion, cell death, mitochondrial respiration, reactive oxygen species production, gene and protein expression, and triacylglycerol accumulation. Culturing HepG2 on collagen I was associated with increased cell proliferation, adhesion, and expression of integrin receptors, and improved cellular spreading compared to culturing them in the absence of collagen I. However, PA-induced lipotoxicity was greater in collagen I-cultured HepG2 than in those cultured in the absence of collagen I and was associated with increased α2β1 receptors. In summary, the present study demonstrated for the first time that collagen I-cultured HepG2 exhibited exacerbated cell death following exposure to PA through integrin-mediated death. The findings from this study may serve as a caution to those using 2D models or 3D scaffold-based models of HepG2 in the presence of collagen I.

• MeSH
• Cell Adhesion * drug effects   MeSH
• Cell Death drug effects   MeSH
• Hep G2 Cells MeSH
• Integrin alpha2beta1 metabolism   MeSH
• Integrins metabolism   genetics   MeSH
• Collagen Type I * metabolism   genetics   MeSH
• Humans MeSH
• Non-alcoholic Fatty Liver Disease metabolism   pathology   MeSH
• Palmitates toxicity   pharmacology   MeSH
• Cell Proliferation * drug effects   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Cell Survival * drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

MicroRNA hsa-miR-29 was connected to a number of malignancies. Its target genes are many, among them Mcl-1 that is expressed in three possible isoforms, one of which is anti-apoptotic and another one pro-apoptotic. Ratio of these two isoforms appears to affect cell response to external stimuli. We have demonstrated that miR-29b enhanced etoposide toxicity in HeLa cell line by modulating this ratio of Mcl-1 isoforms. However, it is not known whether the described miR-29 effect is common to various cancer types or even have the opposite effect. This represents a significant problem for possible future applications. In this report, we demonstrate that miR-29b affects toxicity of 60 μM etoposide in cell lines derived from selected malignancies. The mechanism, however, differs among the cell lines tested. Hep G2 cells demonstrated similar effect of miR-29b on etoposide toxicity as was described in HeLa cells, i.e. modulation of Mcl-1 expression. Target protein down-regulated by miR-29b resulting in enhanced etoposide toxicity in Caco-2 cells was, however, Bcl-2 protein. Moreover, H9c2, Hek-293 and ARPE-19 cell lines selected as a representatives of non-malignant cells, showed no effect of miR-29b on etoposide toxicity. Our data suggest that miR-29b could be a common enhancer of etoposide toxicity in malignant cells due to its modulation of Bcl family proteins.

• MeSH
• Apoptosis drug effects   genetics   MeSH
• Hep G2 Cells MeSH
• Caco-2 Cells MeSH
• Etoposide * toxicity   pharmacology   MeSH
• Antineoplastic Agents, Phytogenic pharmacology   toxicity   MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Humans MeSH
• MicroRNAs * genetics   metabolism   MeSH
• Myeloid Cell Leukemia Sequence 1 Protein * genetics   metabolism   MeSH
• Proto-Oncogene Proteins c-bcl-2 genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Several novel copper (II) complexes of reduced Schiff bases containing fluoride substituents were prepared and structurally characterized by single-crystal X-ray diffraction. The complexes exhibited diverse structures, with the central atom in distorted tetrahedral geometry. The biological effects of the products were evaluated, specifically their cytotoxicity, antimicrobial, and antiurease activities, as well as affinity for albumin (BSA) and DNA (ct-DNA). The complexes showed marked cytotoxic activities in the HepG2 hepatocellular carcinoma cell line, considerably higher than the standard cisplatin. The cytotoxicity depended significantly on the substitution pattern. The best activity was observed in the complex with a trifluoromethyl group in position 4 of the benzene ring-the dichloro[(±)-trans-N,N'-bis-(4-trifluoromethylbenzyl)-cyclohexane-1,2-diamine]copper (II) complex, whose activity (IC50 28.7 μM) was higher than that of the free ligand and markedly better than the activity of the standard cisplatin (IC50 336.8 μM). The same complex also showed the highest antimicrobial effect in vitro. The affinity of the complexes towards bovine serum albumin (BSA) and calf thymus DNA (ct-DNA) was established as well, indicating only marginal differences between the complexes. In addition, all complexes were shown to be excellent inhibitors of the enzyme urease, with the IC50 values in the lower micromolar region.

• MeSH
• Hep G2 Cells MeSH
• DNA metabolism   chemistry   MeSH
• Fluorine chemistry   MeSH
• Coordination Complexes * pharmacology   chemistry   chemical synthesis   MeSH
• Humans MeSH
• Ligands MeSH
• Copper * chemistry   MeSH
• Antineoplastic Agents * pharmacology   chemistry   MeSH
• Schiff Bases * chemistry   pharmacology   MeSH
• Serum Albumin, Bovine chemistry   MeSH
• Urease antagonists & inhibitors   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH