Dynamic Interplay in Tumor Ecosystems: Communication between Hepatoma Cells and Fibroblasts.

Tumors are intricate ecosystems where cancer cells and non-malignant stromal cells, including cancer-associated fibroblasts (CAFs), engage in complex communication. In this study, we investigated the interaction between poorly (HLE) and well-differentiated (HuH7) hepatoma cells and LX2 fibroblasts. We explored various communication channels, including soluble factors, metabolites, extracellular vesicles (EVs), and miRNAs. Co-culture with HLE cells induced LX2 to produce higher levels of laminin ß1, type IV collagen, and CD44, with pronounced syndecan-1 shedding. Conversely, in HuH7/LX2 co-culture, fibronectin, thrombospondin-1, type IV collagen, and cell surface syndecan-1 were dominant matrix components. Integrins α6ß4 and α6ß1 were upregulated in HLE, while α5ß1 and αVß1 were increased in HuH7. HLE-stimulated LX2 produced excess MMP-2 and 9, whereas HuH7-stimulated LX2 produced excess MMP-1. LX2 activated MAPK and Wnt signaling in hepatoma cells, and conversely, hepatoma-derived EVs upregulated MAPK and Wnt in LX2 cells. LX2-derived EVs induced over tenfold upregulation of SPOCK1/testican-1 in hepatoma EV cargo. We also identified liver cancer-specific miRNAs in hepatoma EVs, with potential implications for early diagnosis. In summary, our study reveals tumor type-dependent communication between hepatoma cells and fibroblasts, shedding light on potential implications for tumor progression. However, the clinical relevance of liver cancer-specific miRNAs requires further investigation.

Autophagy may contribute to the recovery of rat mesothelium following acute inflammation in vivo.

Following Freund's adjuvant-induced acute inflammation, the regeneration of rat mesothelium is accompanied by the reduction of cell organelles. The aim of the present study is to test whether autophagy may play a role in the recovery process of mesothelial cells by eliminating accumulated cell organelles and also to investigate the presence of potential inducers and molecular transmitters of the process. Control and treated (from day 2 to day 11; D2-D11) mesothelial cells (n = 16 samples/group) obtained from male rats were isolated and phenotypically characterized. Morphological studies included light and electron microscopy. Biochemical studies performed on tissue samples as well as isolated cells were used to evaluate the dynamics of autophagy and also to detect the expression levels of TNF-α, LC3B, estrogen receptors (ER-α and GPR30) and Erk1/2. Gene expression was measured by individual Taqman assays on quantitative RT-PCR. Protein expression study was performed by Western blotting and immunolabeling. Estradiol concentration was measured both in peritoneal fluid and plasma samples in control and treated animals (n = 3-10 animals per group). Our conventional electron microscopic and morphometric results showed a progressive autophagosome formation with a peak by the termination of inflammation (D5). Subsequently, autophagolysosome formation dominated between D6 and D8 with a concomitant expression of LC3B proved by immunoblotting. We further observed the reduction of cell compartments by D11 parallel with the morphological restitution of mesothelium. Estradiol showed a sustained level in the peritoneal fluid but not in plasma samples between D3 and D11 compared to levels obtained from untreated animals. The mRNA expression of TNF-α was increased between D2 and D11 compared to control. Western blot analysis showed a constitutive expression of GPR30, while ER-α could not be detected between D6 and D11. Erk1/2 was activated by phosphorylation with a peak at D6. Considering our present in vivo results, we hypothesize that the facilitated autophagy might play an important role in the removal of cytoplasmic organelles during the recovery of mesothelium, while our results also suggest that the detected peritoneal estradiol as well as TNF-α may contribute to this process.

BMP-induced non-canonical signaling is upregulated during autophagy-mediated regeneration in inflamed mesothelial cells.

Previously, we showed that after Freund's adjuvant-induced peritonitis, rat mesothelial cells regain their epithelial phenotype through mesenchymal-epithelial transition (MET) accompanied by autophagy. Since bone morphogenetic proteins (BMPs) are well-known MET-inducers, we were interested in the potential expression of BMPs and BMP-induced pathways. Although mesothelial cells expressed lower amounts of BMP7, its level in the peritoneal cavity and mesothelial synthesis of BMP4 were significantly increased during inflammation. BMPR1A and BMPR2 were also significantly expressed. Expression of transforming growth factor beta-activated kinase (TAK1) and c-Jun NH2-terminal kinases (JNK1-JNK2) were more intense than that of phosphorylated Mothers Against Decapentaplegic homolog 1/5 (p-SMAD1/5), confirming that the non-canonical pathway of BMPs prevailed in our model. JNK signaling through B-cell lymphoma-2 (Bcl-2) can contribute to Beclin-1 activation. We demonstrated that TAK1-JNK-Bcl-2 signaling was upregulated simultaneously with the autophagy-mediated regeneration. A further goal of our study was to prove the regenerative role of autophagy after inflammation. We used a specific inhibitor, bafilomycin A1 (BafA1), and found that BafA1 treatment decreased the expression of microtubule-associated protein 1A/1B-light chain 3 (LC3B) and resulted in morphological signs of cell death in inflamed mesothelial cells indicating that if autophagy is arrested, regeneration turns into cell death and consequently, mesothelial cells die.

Inflammation in Focus: The Beginning and the End.

The inflammation is an important biological response induced by various harmful stimuli, like viruses, bacterial infections, toxins, toxic compounds, tissue injury. During inflammation inflammatory cytokines and reactive oxygen species are produced. Inflammatory cytokines act on various receptors present on the plasma membrane of target cells. To initiate signaling cascade, and activate transcription factors, receptors should be internalized and enter the early endosomes, where the members of the signaling cascade can meet. The further cytoplasmic fate of the receptor plays crucial role in the progression and the course of inflammation. Usually acute inflammation removes injurious stimuli and helps to regain the normal healthy status of the organism. In contrast to this the uncontrolled chronic inflammation-stimulating other than immune cells, inducing transdifferentiation-can provide base of various serious diseases. This paper draws the attention of the long-lasting consequence of chronic inflammation, pointing out that one of the most important step in medication is to identify in time the factors initiating and maintaining inflammation.

Preconditioning with cell-free DNA prevents DSS-colitis by promoting cell protective autophagy.

Presence of cell-free DNA (cfDNA) in sera of patients with inflammatory bowel diseases (IBD) is a long-known fact. The biological effect of cfDNA administration on cellular autophagy within normal and inflammatory circumstances remains unclear. In this study, the effects of intravenous cfDNA pretreatment on autophagy response were studied in dextran sulfate sodium (DSS)-induced acute experimental colitis. Selected proinflammatory cytokine and autophagy-related gene and protein expressions were compared with clinical and histological activity parameters, and with transmission electron microscopic evaluations. A single intravenous dose of cfDNA pretreatment with cfDNA from colitis exhibited beneficial response concerning the clinical and histological severity of DSS-colitis as compared with effects of normal cfDNA. Pretreatment with colitis-derived cfDNA substantially altered the gene and protein expression of several autophagy and inflammatory cytokine genes in a clinically favorable manner. Autophagy in splenocytes is also altered after colitis-derived cfDNA pretreatment. During the process of acute colitis, the subsequent inflammatory environment presumably results in changes of cfDNA with the potential to facilitate cell protective autophagy. Understanding the molecular mechanisms behind the impact of colitis-associated autophagy, and elucidating alterations of the interaction between autophagy and innate immunity caused by nucleic acids may provide further insight into the etiology of IBD. By targeting or modifying cfDNA, novel anti-inflammatory therapies may be developed.

[The role of caveolae in cataractogenesis: examination of human lens epithelial cells]. / Caveolák szerepe a szürke hályog képzodésében: humán szemlencse epithelsejtjeinek vizsgálata.

Inrtoduction: Caveolae are flask shaped with 50-100 nm size, non-clathrin associated invaginations of the plasmamembrane. The main membrane protein of the structures is caveolin-1. Caveolae play an important role in numerous cellular functions including vesicular transport and cell-cycle regulation, and create platforms for classical and alternative signaling pathways. According to international studies, caveolae may influence the physiology and pathology of lens epithelial cells. AIM: The aim of the study was to examine and compare the morphology of caveolae and the immunohistochemical difference of caveolin-1 in control (myopic and hyperopic) lens epithelial cells and human lens epithelial cells affected by cataract. Authors investigated whether caveolae might have a role in cataractogenesis. METHOD: Anterior lens capsules were obtained by capsulorhexis during surgery of senile cataract and refractive surgery of the clear lens. Ultra-fine sections have been studied by transmission electron microscopy, and semi-fine samples were labelled for immunohistochemistry with polyclonal caveolin-1 and cavin-1 antibodies. RESULTS: By immunohistochemistry, in the control group, significant caveolin-1 label with low cavin-1 signal were measured in the lens epithelial cells. In the cataract group high cavin-1 and caveolin-1 expression was detected. In the control group, caveolae were not observed, but in the lens epithelial cells with cataract, increased number of caveolae have been detected by electron microscopy. CONCLUSIONS: For the development and maintenance of the specific caveolae shape, caveolin-1 is needed to be accompanied by cavin-1. Therefore, it is presumable that the increased expression of cavin-1 could explain the higher number of caveolae in the cataract group. These results might suggest that caveolae might play a role in cataractogenesis. Orv Hetil. 2019; 160(8): 300-308.