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Oncology Reports Jul 2023Lentinan (LNT) isolated from is a vital host defense potentiator previously utilized as an adjuvant in cancer therapy. The present study investigated the effect of LNT...
Lentinan (LNT) isolated from is a vital host defense potentiator previously utilized as an adjuvant in cancer therapy. The present study investigated the effect of LNT on the mouse hepatocellular carcinoma (HCC) cell line Hepa1‑6 and its possible mechanism. Mouse HCC apoptosis and its potential associated mechanism were then explored using and approaches. For approaches, the effect of LNT on the proliferation of Hepa1‑6 cells was investigated by Cell Counting Kit‑8 assay. Annexin V‑FITC staining and flow cytometry were applied to explore HCC apoptosis. Western blotting was used to analyze related proteins, such as EGR1, phosphatase and tensin homolog (PTEN), phosphorylated protein kinase B (p‑Akt), protein kinase B (Akt), B lymphocyte‑2 (Bcl‑2), Bcl2 family‑associated X protein (Bax), etc. Cellular immunofluorescence staining was employed to assess the localization and expression of EGR1 and PTEN in nuclear and cytoplasmic fractions of Hepa1‑6 cells. The association between and was explored by overexpression in cell lines. For methods, a mouse model of diethylnitrosamine (DEN)‑induced primary liver cancer was established using C57BL/6 mice to investigate the inhibitory effect of LNT on liver cancer. Histopathology of liver tissue from mice was detected by hematoxylin‑eosin staining and immunohistochemical assay. and results showed that LNT can inhibit the proliferation and promote the apoptosis of mouse HCC cells. Besides, LNT increased the expression of EGR1 in Hepa1‑6 cells, which is translocated to the nucleus to function as a transcriptional factor. EGR1 then activates the expression of the tumor suppressor PTEN, thereby inhibiting the activation of the AKT signaling pathway. These data revealed a novel anti‑tumor mechanism by which LNT can induce apoptosis to inhibit mouse HCC progression through the EGR1/PTEN/AKT axis. These results provide a scientific basis for the potential use of LNT in drug development and clinical applications associated with primary liver cancer.
Topics: Mice; Animals; Proto-Oncogene Proteins c-akt; Carcinoma, Hepatocellular; Lentinan; Liver Neoplasms; Cell Line, Tumor; Mice, Inbred C57BL; Mice, Inbred Strains; Signal Transduction; Apoptosis; PTEN Phosphohydrolase; Cell Proliferation; Early Growth Response Protein 1
PubMed: 37264970
DOI: 10.3892/or.2023.8579 -
Gastroenterologia Y Hepatologia Apr 2024The leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) plays an important role in stem cell differentiation, organ development and cancer. Whether LGR4...
BACKGROUND
The leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) plays an important role in stem cell differentiation, organ development and cancer. Whether LGR4 affects the progression of hepatocellular carcinoma (HCC) remains unknown. This study aimed to reveal the role of LGR4 in HCC.
METHODS
Clinical samples of HCC were collected to assess the expression of LGR4 and its correlation with patients' clinical characteristics. The expression level of LGR4 in HCC cells was altered by pharmacological and genetic methods, and the role of LGR4 in HCC progression was analyzed by in vivo and in vitro assays. HCC was induced by diethylnitrosamine (DEN) and carbon tetrachloride (CCl) in wild-type and LGR4 deficient mice, the effect of LGR4 on HCC was examined by histopathological evaluation and biochemical assays.
RESULTS
LGR4 expression was up-regulated in HCC samples, and its expression level was positively correlated with tumor size, microvascular invasion (MVI), TNM stage and pathological differentiation grade of HCC patients. In the mouse HCC model induced by DEN+CCl, knockdown of LGR4 effectively inhibited the progression of HCC. Silencing of LGR4 inhibited the proliferation, migration, invasion, stem cell-like properties and Warburg effect of HCC cells. These phenotypes were promoted by R-spondin2 (Rspo2), an endogenous ligand for LGR4. Rspo2 markedly increased the nuclear translocation of β-catenin, whereas IWR-1, an inhibitor of Wnt/β-catenin signaling, reversed its effect. Deficiency of LGR4 significantly reduced the nuclear translocation of β-catenin and the expression of its downstream target genes cyclinD1 and c-Myc.
CONCLUSIONS
LGR4 promotes HCC progression via Wnt/β-catenin signaling pathway.
Topics: Humans; Animals; Mice; Carcinoma, Hepatocellular; Wnt Signaling Pathway; beta Catenin; Liver Neoplasms; Cell Differentiation; Disease Models, Animal; Receptors, G-Protein-Coupled
PubMed: 37437654
DOI: 10.1016/j.gastrohep.2023.05.016 -
Cancers Aug 2023Primary liver cancer (PLC) can be classified in hepatocellular (HCC), cholangiocarcinoma (CCA), and combined hepatocellular-cholangiocarcinoma (cHCC-CCA). The molecular...
Primary liver cancer (PLC) can be classified in hepatocellular (HCC), cholangiocarcinoma (CCA), and combined hepatocellular-cholangiocarcinoma (cHCC-CCA). The molecular mechanisms involved in PLC development and phenotype decision are still not well understood. Complete deletion of encoding the B56δ subunit of Protein Phosphatase 2A (PP2A) results in spontaneous HCC development in mice via a c-MYC-dependent mechanism. In the present study, we aimed to examine the role of in an independent mouse model of diethylnitrosamine (DEN)-induced hepatocarcinogenesis. deletion (heterozygous and homozygous) accelerated HCC development, corroborating its tumor-suppressive function in liver and suggesting may be haploinsufficient. -deficient HCCs stained positively for c-MYC, consistent with increased AKT activation in pre-malignant and tumor tissues of -deficient mice. We also found increased YAP activation in -deficient tumors. Remarkably, in older mice, deletion resulted in cHCC-CCA development in this model, with the CCA component showing increased expression of progenitor markers (SOX9 and EpCAM). Finally, we observed an upregulation of in tumors from wildtype and heterozygous mice, revealing a tumor-specific control mechanism of expression, and suggestive of the involvement of in a negative feedback regulation restricting tumor growth. Our study highlights the tumor-suppressive role of mouse PP2A-B56δ in both HCC and cHCC-CCA, which may have important implications for human PLC development and targeted treatment.
PubMed: 37627221
DOI: 10.3390/cancers15164193 -
The Journal of Biological Chemistry Nov 2023Wilm's tumor 1-associating protein (WTAP), a regulatory protein of the mA methyltransferase complex, has been found to play a role in regulating various physiological...
Wilm's tumor 1-associating protein (WTAP), a regulatory protein of the mA methyltransferase complex, has been found to play a role in regulating various physiological and pathological processes. However, the in vivo role of WTAP in the pathogenesis of hepatocellular carcinoma (HCC) is unknown. In this study, we have elucidated the crucial role of WTAP in HCC progression and shown that hepatic deletion of Wtap promotes HCC pathogenesis through activation of multiple signaling pathways. A single dose of diethylnitrosamine injection causes more and larger HCCs in hepatocyte-specific Wtap knockout (Wtap-HKO) mice than Wtap mice fed with either normal chow diet or a high-fat diet. Elevated CD36, IGFBP1 (insulin-like growth factor-binding protein 1), and chemokine (C-C motif) ligand 2 (CCL2) expression leads to steatosis and inflammation in the Wtap-HKO livers. The hepatocyte proliferation is dramatically increased in Wtap-HKO mice, which is due to higher activation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription-3 signaling pathways. Hepatic deletion of Wtap activates the ERK signaling pathway by increasing the protein stability of GRB2 and ERK1/2, which is due to the decreased expression of proteasome-related genes. Restoring PSMB4 or PSMB6 (two key components of the proteasome) leads to the downregulation of GRB2 and ERK1/2 in Wtap-HKO hepatocytes. Mechanistically, WTAP interacts with RNA polymerase II and H3K9ac to maintain expression of proteasome-related genes. These results demonstrate that hepatic deletion of Wtap promotes HCC progression through activating GRB2-ERK1/2-mediated signaling pathway depending on the downregulation of proteasome-related genes especially Psmb4 and Psmb6.
Topics: Animals; Mice; Carcinoma, Hepatocellular; Cell Proliferation; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Hepatocytes; Liver Neoplasms; Mice, Knockout; Proteasome Endopeptidase Complex; Mice, Inbred C57BL
PubMed: 37777158
DOI: 10.1016/j.jbc.2023.105301 -
International Journal of Molecular... Jul 2023Inflammatory processes play major roles in carcinogenesis and the progression of hepatocellular carcinoma (HCC) derived from non-alcoholic steatohepatitis (NASH). But,...
Inflammatory processes play major roles in carcinogenesis and the progression of hepatocellular carcinoma (HCC) derived from non-alcoholic steatohepatitis (NASH). But, there are no therapies for NASH-related HCC, especially focusing on these critical steps. Previous studies have reported that farnesyltransferase inhibitors (FTIs) have anti-inflammatory and anti-tumor effects. However, the influence of FTIs on NASH-related HCC has not been elucidated. In hepatoblastoma and HCC cell lines, HepG2, Hep3B, and Huh-7, we confirmed the expression of hypoxia-inducible factor (HIF)-1α, an accelerator of tumor aggressiveness and the inflammatory response. We established NASH-related HCC models under inflammation and free fatty acid burden and confirmed that HIF-1α expression was increased under both conditions. Tipifarnib, which is an FTI, strongly suppressed increased HIF-1α, inhibited cell proliferation, and induced apoptosis. Simultaneously, intracellular interleukin-6 as an inflammation marker was increased under both conditions and significantly suppressed by tipifarnib. Additionally, tipifarnib suppressed the expression of phosphorylated nuclear factor-κB and transforming growth factor-β. Finally, in a NASH-related HCC mouse model burdened with diethylnitrosamine and a high-fat diet, tipifarnib significantly reduced tumor nodule formation in association with decreased serum interleukin-6. In conclusion, tipifarnib has anti-tumor and anti-inflammatory effects in a NASH-related HCC model and may be a promising new agent to treat this disease.
Topics: Mice; Animals; Carcinoma, Hepatocellular; Non-alcoholic Fatty Liver Disease; Liver Neoplasms; Farnesyltranstransferase; Interleukin-6; Hypoxia-Inducible Factor 1, alpha Subunit; Enzyme Inhibitors; Anti-Inflammatory Agents; Inflammation; Cell Line, Tumor
PubMed: 37511305
DOI: 10.3390/ijms241411546 -
Science Advances Feb 2024Physiologically, FoxA1 plays a key role in liver differentiation and development, and pathologically exhibits an oncogenic role in prostate and breast cancers. However,...
Physiologically, FoxA1 plays a key role in liver differentiation and development, and pathologically exhibits an oncogenic role in prostate and breast cancers. However, its role and upstream regulation in liver tumorigenesis remain unclear. Here, we demonstrate that FoxA1 acts as a tumor suppressor in liver cancer. Using a CRISPR-based kinome screening approach, noncanonical inflammatory kinase IKBKE has been identified to inhibit FoxA1 transcriptional activity. Notably, IKBKE directly binds to and phosphorylates FoxA1 to reduce its complex formation and DNA interaction, leading to elevated hepatocellular malignancies. Nonphosphorylated mimic knock-in mice markedly delay liver tumorigenesis in hydrodynamic transfection murine models, while phospho-mimic knock-in phenocopy knockout mice to exhibit developmental defects and liver inflammation. Notably, knockout delays diethylnitrosamine (DEN)-induced mouse liver tumor development. Together, our findings not only reveal FoxA1 as a bona fide substrate and negative nuclear effector of IKBKE in hepatocellular carcinioma (HCC) but also provide a promising strategy to target IKBEK for HCC therapy.
Topics: Animals; Male; Mice; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Hepatocyte Nuclear Factor 3-alpha; Liver Neoplasms; Mice, Knockout
PubMed: 38324694
DOI: 10.1126/sciadv.adk2285 -
The Biochemical Journal Nov 2023Staphylococcal nuclease Tudor domain containing 1 (SND1) protein is an oncogene that 'reads' methylarginine marks through its Tudor domain. Specifically, it recognizes...
Staphylococcal nuclease Tudor domain containing 1 (SND1) protein is an oncogene that 'reads' methylarginine marks through its Tudor domain. Specifically, it recognizes methylation marks deposited by protein arginine methyltransferase 5 (PRMT5), which is also known to promote tumorigenesis. Although SND1 can drive hepatocellular carcinoma (HCC), it is unclear whether the SND1 Tudor domain is needed to promote HCC. We sought to identify the biological role of the SND1 Tudor domain in normal and tumorigenic settings by developing two genetically engineered SND1 mouse models, an Snd1 knockout (Snd1 KO) and an Snd1 Tudor domain-mutated (Snd1 KI) mouse, whose mutant SND1 can no longer recognize PRMT5-catalyzed methylarginine marks. Quantitative PCR analysis of normal, KO, and KI liver samples revealed a role for the SND1 Tudor domain in regulating the expression of genes encoding major acute phase proteins, which could provide mechanistic insight into SND1 function in a tumor setting. Prior studies indicated that ectopic overexpression of SND1 in the mouse liver dramatically accelerates the development of diethylnitrosamine (DEN)-induced HCC. Thus, we tested the combined effects of DEN and SND1 loss or mutation on the development of HCC. We found that both Snd1 KO and Snd1 KI mice were partially protected against malignant tumor development following exposure to DEN. These results support the development of small molecule inhibitors that target the SND1 Tudor domain or the use of upstream PRMT5 inhibitors, as novel treatments for HCC.
Topics: Animals; Mice; Carcinoma, Hepatocellular; Endonucleases; Liver Neoplasms; Nuclear Proteins; Transcription Factors; Genetic Predisposition to Disease
PubMed: 37905668
DOI: 10.1042/BCJ20230384 -
The Yale Journal of Biology and Medicine Dec 2023Identifying new hepatocellular carcinoma (HCC)-driven signaling molecules and discovering their molecular mechanisms are crucial for efficient and better outcomes....
Identifying new hepatocellular carcinoma (HCC)-driven signaling molecules and discovering their molecular mechanisms are crucial for efficient and better outcomes. Recently, OMA1 and YME1L, the inner mitochondrial proteases, were displayed to be associated with tumor progression in various cancers; however, their role in HCC has not yet been studied. Therefore, we evaluated the possible role of OMA1/YME1L in HCC staging and discussed their potential role in cellular apoptosis and proliferation. Our study was performed using four groups of male albino rats: a normal control and three diethyl nitrosamine-treated groups for 8, 16, and 24 weeks. The OMA1 and YME1L, matrix-metalloproteinase-9 (MMP-9), and cyclin D1 content were measured in liver tissues, while alpha-fetoprotein (AFP) level was assessed in serum. Additionally, Ki-67 expression was evaluated by immunohistochemistry. The relative hepatic expression of Bax, and tissue inhibitor matrix metalloproteinase (TIMP-3) was measured. Herein, we confirmed for the first time that OMA1 is down-regulated while YME1L is up-regulated in HCC in the three studied stages with subsequent inhibition of apoptosis and cell cycle progression. Furthermore, these proteases have a possible role in metastasis. These newly recognized results suggested OMA1 and YME1L as possible diagnostic tools and therapeutic targets for HCC management.
Topics: Male; Animals; Rats; Diethylnitrosamine; Metalloproteases; Mitochondrial Proteins; Carcinoma, Hepatocellular; Liver Neoplasms; Neoplasm Staging; ATPases Associated with Diverse Cellular Activities; Apoptosis; Neoplasm Metastasis; Oxidative Stress; Liver; Biomarkers, Tumor
PubMed: 38161580
DOI: 10.59249/BWBY8971 -
BioRxiv : the Preprint Server For... Nov 2023Precision-Cut Liver Slices (PCLS) are an culture model developed to study hepatic drug metabolism. One of the main benefits of this model is that it retains the...
BACKGROUND
Precision-Cut Liver Slices (PCLS) are an culture model developed to study hepatic drug metabolism. One of the main benefits of this model is that it retains the structure and cellular composition of the native liver. PCLS also represents a potential model system to study liver fibrosis in a setting that more closely approximates pathology than methods. The aim of this study was to assess whether responses to antifibrotic interventions can be detected and quantified with PCLS.
METHODS
PCLS of 250 μm thickness were prepared from four different murine fibrotic liver models: choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD), thioacetamide (TAA), diethylnitrosamine (DEN), and carbon tetrachloride (CCl). PCLS were treated with 5 μM Erlotinib for 72 hours. Histology and gene expression were then compared with murine experiments and TGF-β1 activated hepatic stellate cells (HSCs). These types of PCLS characterization were also evaluated in PCLS from human cirrhotic liver.
RESULTS
PCLS viability in culture was stable for 72 hours. Treatment of erlotinib, an EGFR inhibitor significantly inhibited the expression of profibrogenic genes and in PCLS from CDAHFD-induced cirrhotic mice, and and in PCLS from TAA-induced cirrhotic rats. Erlotinib treatment of PCLS from DEN-induced cirrhotic rats inhibited the expression of and , which was consistent with the impact of erlotinib on and expression in DEN-induced cirrhosis. Erlotinib treatment of PCLS from CCl-induced cirrhosis caused reduced expression of and , which was consistent with the effect of erlotinib in CCl-induced cirrhosis. In addition, in HSCs at PCLS from normal mice, TGF-β1 treatment upregulated (), while treatment with erlotinib inhibited the expression of . Similar expression results were observed in TGF-β1 treated HSCs. Expression of MMPs and TIMPs, key regulators of fibrosis progression and regression, were also significantly altered under erlotinib treatment in PCLS. Expression changes under erlotinib treatment were also corroborated with PCLS from human cirrhosis samples.
CONCLUSION
The responses to antifibrotic interventions can be detected and quantified with PCLS at the gene expression level. The antifibrotic effects of erlotinib are consistent between PCLS models of murine cirrhosis and those observed and . Similar effects were also reproduced in PCLS derived from patients with cirrhosis. PCLS is an excellent model to assess antifibrotic therapies that is aligned with the principles of Replacement, Reduction and Refinement (3Rs).
PubMed: 37961334
DOI: 10.1101/2023.10.30.564772 -
Frontiers in Immunology 2023Liver cancers exhibit abnormal (leaky) vasculature, hypoxia and an immunosuppressive microenvironment. Normalization of tumor vasculature is an emerging approach to...
INTRODUCTION
Liver cancers exhibit abnormal (leaky) vasculature, hypoxia and an immunosuppressive microenvironment. Normalization of tumor vasculature is an emerging approach to treat many cancers. Blockmir CD5-2 is a novel oligonucleotide-based inhibitor of the miR-27a interaction with VE-Cadherin, the endothelial-specific cadherin. The combination of a vasoactive medication with inhibition of immune checkpoints such as programmed cell death protein 1 (PD1) has been shown to be effective in treating liver cancer in humans. We aimed to study the effect of CD5-2 combined with checkpoint inhibition (using an antibody against PD1) on liver tumor growth, vasculature and immune infiltrate in the diethylnitrosamine (DEN)-induced liver tumor mouse model.
METHODS
We first analyzed human miR-27a and VE-Cadherin expression data from The Cancer Genome Atlas for hepatocellular carcinoma. CD5-2 and/or anti-PD1 antibody were given to the DEN-treated mice from age 7-months until harvest at age 9-months. Tumor and non-tumor liver tissues were analyzed using histology, immunohistochemistry, immunofluorescence and scanning electron microscopy.
RESULTS
Human data showed high miR-27a and low VE-Cadherin were both significantly associated with poorer prognosis. Mice treated with CD5-2 plus anti-PD1 antibody had significantly smaller liver tumors (50% reduction) compared to mice treated with either agent alone, controls, or untreated mice. There was no difference in tumor number. Histologically, tumors in CD5-2-treated mice had less leaky vessels with higher VE-Cadherin expression and less tumor hypoxia compared to non-CD5-2-treated mice. Only tumors in the combination CD5-2 plus anti-PD1 antibody group exhibited a more favorable immune infiltrate (significantly higher CD3+ and CD8+ T cells and lower Ly6G+ neutrophils) compared to tumors from other groups.
DISCUSSION
CD5-2 normalized tumor vasculature and reduced hypoxia in DEN-induced liver tumors. CD5-2 plus anti-PD1 antibody reduced liver tumor size possibly by altering the immune infiltrate to a more immunosupportive one.
Topics: Humans; Mice; Animals; Infant; Diethylnitrosamine; Liver Neoplasms; MicroRNAs; Hypoxia; Tumor Microenvironment
PubMed: 37795103
DOI: 10.3389/fimmu.2023.1245708