Hepatoma Cells Research Articles

Wogonin inhibits radiation-induced DNA damage repair in hepatocellular carcinoma cells by upregulating p21.

Wogonin has been shown to exhibit anti-tumor effects by regulating the growth and inducing cell death in hepatocellular carcinoma (HCC) cells. However, its impact on radiotherapy for HCC remains unclear. This study aimed to elucidate the mechanisms and effects of wogonin in enhancing radiotherapy for HCC. The viability and cell cycle of HCC cells were assessed using CCK-8, trypan blue dye exclusion, and flow cytometry. RNA sequencing was performed to explore the genomic effects of wogonin on HCC cells. Immunofluorescence staining was employed to detect γ-H2AX distribution, and Western blot was used to evaluate the expression of γ-H2AX and p21. Wogonin induced cell cycle arrest and inhibited DNA damage repair in SMMC-7721 and HCC-LM3 cells following irradiation. RNA sequencing analysis of wogoninand radiation-treated cells revealed significant enrichment of genes related to cell cycle progression, with notable changes in CDK inhibitor expression. Furthermore, wogonin in combination with irradiation increased the expression of γ-H2AX and p21 in HCC cells. Notably, p21 interference partially abrogated the anti-tumor effects of wogonin and radiation. Wogonin enhances the efficacy of radiotherapy in HCC by promoting cell cycle arrest and inhibiting DNA damage repair through upregulation of p21.

GP73-mediated secretion of PKM2 and GP73 promotes angiogenesis and M2-like macrophage polarization in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. Abnormally high expression of Golgi protein 73 (GP73) and pyruvate kinase M2 (PKM2) is intimately associated with HCC progression. However, as secreted proteins, the role of their extracellular secretions in HCC progression remains unclear. Here, we demonstrated that the expression of extracellular GP73 was positively correlated with extracellular PKM2. GP73 interacted with PKM2 to promote SUMO1 modification of PKM2, which in turn enhanced the interaction of GP73 and PKM2. This process continuously promoted the transfer of PKM2 from the cytoplasm to the membrane in HCC cells, and finally secretion. Extracellular PKM2 and GP73 synergistically promoted angiogenesis and polarization of M2-type macrophages, thereby leading to malignant progression and sorafenib resistance in HCC. Sorafenib combined with shikonin, a specific inhibitor of PKM2, has a strong anti-tumor effect. This study reveals the role of GP73 in enhancing PKM2 and GP73 secretion in promoting HCC progression, providing a theoretical basis and drug targets for HCC therapy.

UHPLC‐QTOF‐MS‐Based Metabolic Profiling, Estimation of Antioxidants and Anticancer Potential Assessment of Dicliptera bupleuroides Nees by Caspase Activation, and ROS and MMP Evaluation by Using Fluorescence Microscopy

ABSTRACTDicliptera bupleuroides has been used traditionally for treating various ailments in many countries. Its detailed phytochemical profiling and pharmacological evaluation in treating cancer based on reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) analysis along with caspase activation studies has not been done previously. This study was conducted for detailed phytochemical profiling of D. bupleuroides by using ultra‐high performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UHPLC‐QTOF‐MS) and to evaluate its antioxidant and anticancer potential against hepatocellular carcinoma (HepG2) cells. The extract was subjected to UHPLC‐QTOF‐MS analysis for metabolite profiling, and GNPS Molnet enhancer determined its major phytochemical classes. Phytochemical tests and multimode antioxidant assays were also executed. The extract was pharmacologically evaluated for its anticancer potential against HepG2 cells using CCK‐8 assay. Intracellular ROS generation and loss in MMP were studied by fluorescence microscopy. Network pharmacology and molecular docking of selected compounds were executed. The extract contains total phenolic content as 58.37 ± 0.32 μg GAE/mg extract, and remarkable antioxidant potential was revealed in total antioxidant contents (TAC) and total reducing power (TRP) assays, i.e., 70.4 ± 0.4, 83.2 ± 1.9 μg ascorbic acid equilant (AAE) Borges/mg extract. The UHPLC‐QTOF‐MS analysis showed that the extract contains flavonoid glycosides, phenolic acids, flavans, O‐methylated flavonoids, linoleic acids, terpene glycosides, triterpenoid saponins, and certain other phytoconstituents. Maximum inhibition of 77.13% ± 1.60% against HepG2 cells was observed at 24 h. Loss in MMP by JC‐1 (5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimi‐ dazolylcarbocyanine iodide) staining and increased ROS generation, and caspase‐3, caspase‐8 activation by extract significantly illustrated the cancer cell death by apoptosis. D. bupleuroides could be a potential source of safe and therapeutically active compounds that can be purified to develop safer, selective, and efficacious anticancer agents with fewer side effects.

Positively Charged Nanoplastics Destruct the Structure of the PCK1 Enzyme, Promote the Aerobic Gycolysis Pathway, and Induce Hepatic Tumor Risks.

The production and weathering processes of nanoparticles (NPs) introduce charged functional groups on their surface. Previous studies have found that the surface charge properties of NPs play a critical role in their toxic effects and the mechanisms are generally attributed to their different accumulations and transmembrane potentials. Currently, we still lack sufficient knowledge about effects, owing to the unique structures of these polymers. In this study, positively charged NPs (PS-NH2, 50 nm) at 0.05-0.5 μg mL-1 promoted the proliferation of hepatic tumors in oncogenic KrasG12V zebrafish larvae and they also increased the viability of human hepatocellular carcinoma cells, whereas negatively charged NPs (PS-COOH, 50 nm) did not. We present evidence indicating that the potential carcinogenicity of PS-NH2 is related to the special polymer-molecular interactions caused by its positive surface charge. The affinity of PS-COOH chains for peptides typically enhances enzyme stability and upregulates its expression. However, PS-NH2 strongly competes with hydrogen bonds of the first rate-limiting enzyme PCK1 in gluconeogenesis, thus downregulating the expression of PCK1 and promoting the aerobic glycolysis pathway, which most tumor cells prefer. This study indicates that positive-charge modified NPs in the environment may bring additional carcinogenic risks.

The Role of Glutamine Synthetase on the Sensitivity to Radiotherapy of Hepatocellular Carcinoma.

The objective of this study was to investigate the relationship between radiotherapy sensitivity, glutamine synthetase (GS), and oxidative stress (OS) in human hepatocellular carcinoma (HCC) cells. HCC cells were X-ray irradiated, and the effect of glutamine synthetase inhibition on the proliferative capacity of HCC cells was examined using the CCK-8 colony formation assay. Real-time quantitative PCR assays were used to detect the effect of L-methionine sulfoximine (MSO) on cellular glutamine synthetase expression levels and the efficiency of glutamine synthetase knockdown in HepG2 cells. Glutamine synthetase activity assay kit was used to detect the viability of glutamine synthetase in cells and tissues. Oxidative stress production was assayed using an oxidative stress assay kit. Subcutaneous xenografts were used to detect the effects of L-methionine sulfoximine and radiation on tumor growth invivo. The results showed that the apparent cell proliferation capacity of HCC cells after glutamine synthetase inhibition was significantly reduced after radiotherapy, which was closely related to the increased production of oxidative stress after radiotherapy. Furthermore, the results of animal experiments also showed that the combination of L-methionine sulfoximine and radiation induced a stronger tumor suppressive effect and that L-methionine sulfoximine could act as a radiosensitizer after radiotherapy.

Identifying potential signatures of immune cells in hepatocellular carcinoma using integrative bioinformatics approaches and machine-learning strategies.

Hepatocellular carcinoma (HCC) is a malignant tumor regulated by the immune system. Immunotherapy using checkpoint inhibitors has shown encouraging outcomes in a subset of HCC patients. The main challenges in checkpoint immunotherapy for HCC are to expand treatment options and to broaden the beneficiary population. Therefore, the search for potential signatures of immune cells is meaningful in the development of immunotherapy for HCC. The HCC related datasets were downloaded from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). Differential expression analysis and functional analysis were performed first. Then support vector machine-recursive feature elimination (SVM-RFE), random forests (RF), least absolute shrinkage and selection operation (LASSO), and weighed gene co-expression network analysis (WGCNA) were employed to screen for critical genes, and receiver operating characteristic (ROC) analysis was performed to compare diagnostic performance. Subsequently, single-sample gene set enrichment analysis (ssGSEA) was used to explore the relationship between signatures and immune cells. Finally, we validated the expression of these biomarkers in human HCC samples. 531 overlapping differentially expressed genes (DEGs) were identified. Furthermore, enrichment analysis revealed pathways associated with immune activation processes, immune cell involvement and inflammatory signaling. After using multiple machine-learning strategies, extracellular matrix protein 1 (ECM1), leukemia inhibitory factor receptor (LIFR), sushi repeat containing protein X-linked (SRPX), and thromboxane A2 receptor (TBXA2R) were identified as critical signatures, and exhibited high expression in tumor-adjacent normal tissues. According to the ssGSEA results, ECM1, LIFR, SRPX and TBXA2R were all significantly associated with diverse immune cells, such as monocytes and neutrophils. Moreover, immunostaining of human HCC samples showed that these critical signatures all colocalized with CD14-positive monocytes. Our findings report the potential signatures of immune cells in HCC and confirm that they localize in monocytes of tumor-adjacent normal tissues. ECM1, LIFR, SRPX and TBXA2R could become new potential targets for predictive diagnosis, early intervention and immunotherapy of HCC in the future.

Silencing LINC01547 induces hepatocellular carcinoma cell apoptosis and metastasis inhibition via the ADAR1/FAK and miR-146b-5p/RAC1 axes.

Growing research indicates that long noncoding RNAs (lncRNAs) are pivotal in the development and advancement of hepatocellular carcinoma (HCC). Our research pinpointed LINC01547 as a notable lncRNA that was significantly downregulated in Hep3B cells treated with bufotalin, whereas it exhibited elevated expression levels in HCC tumor tissues. Further study found that silencing LINC01547 markedly suppressed proliferation, induced apoptosis, and inhibited migration and invasion in Hep3B and HepG2 cells. LINC01547 knockdown reduced ADAR1 expression, which led to apoptosis and suppressed metastasis via inhibition of the FAK signaling pathway. Additionally, silencing LINC01547 upregulated miR-146b-5p, which in turn decreased RAC1 levels, further promoting apoptosis and inhibiting metastasis in HCC cells. In vivo, a Hep3B tumor-bearing mouse model confirmed the antitumor effects of LINC01547 silencing. Our findings demonstrate that LINC01547 regulates HCC cell apoptosis and metastasis through the ADAR1/FAK and miR-146b-5p/RAC1 pathways, suggesting that LINC01547 may serve as a biomarker and potential therapeutic target for HCC.

Antitumor effects of natural killer cells derived from gene-engineered human-induced pluripotent stem cells on hepatocellular carcinoma.

Mortality and recurrence rates of hepatocellular carcinoma (HCC) remain high despite the use of various treatment methods. Recently, cell-based immunotherapy using natural killer (NK) cells has attracted considerable attention in cancer immunotherapy. NK cells generated from induced pluripotent stem cells (iPSCs) are a new option for use as an NK cell resource. The eNK cells (HLCN061, developed by HEALIOS K.K.) are human iPSC-derived NK cells differentiated from clinical-grade iPSCs in which IL-15, CCR2B, CCL19, CD16a, and NKG2D have been introduced. In this study, we aimed to evaluate the potential of eNK cell therapy for HCC treatment. The analysis of eNK cells for cell surface and intracellular molecules revealed that antitumor-related surface molecules (TRAIL, CD226, and CD16) and intracellular cytotoxic factors (perforin, granzyme B, TNFα, and IFNγ) were highly expressed. In addition, eNK cells exhibited high cytotoxicity against HCC cell lines (HepG2, HuH7, and SNU-423), which are sensitive to NKG2D, TRAIL, and CD226. The TRAIL and perforin/granzyme B pathways are largely involved in this cytotoxic mechanism, as indicated by the reduction in cytotoxicity induced by TRAIL inhibitory antibodies and concanamycin A, which inhibits perforin/granzyme B-mediated cytotoxicity. Our data suggest that eNK cells, whose functions have been enhanced by genetic engineering, have the potential to improve HCC treatment.

Machine learning-based integration reveals immunological heterogeneity and the clinical potential of T cell receptor (TCR) gene pattern in hepatocellular carcinoma.

The T Cell Receptor (TCR) significantly contributes to tumor immunity, whereas the intricate interplay with the Hepatocellular Carcinoma (HCC) microenvironment and clinical significance remains largely unexplored. Here, we aimed to examine the function of TCR signaling in tumor immunity and its clinical significance in HCC. Our objective was to employ TCR signaling genes and a machine learning-based integrative methodology to construct a prognostic prediction system termed the TCR score. Herein, we revealed that the TCR score serves as an independent risk factor for overall survival in HCC patients, demonstrating stable and robust performance. The accuracy of the TCR score significantly exceeds that of traditional clinical variables and published signatures. Additionally, the immune infiltration was abundant in patients with low TCR scores. Single-cell cohort analysis further demonstrates that patients with low TCR scores possess an immune-active tumor microenvironment (TME), with T/NK cells enhancing interactions with myeloid cells through signaling networks such as MIF, MK, and SPP1. In response to these changes in the TME, patients with high TCR scores exhibit poorer outcomes and shorter survival in immunotherapy cohorts. In vitro experiments demonstrated that the key TCR signaling biomarker SOS1 knockdown significantly suppresses the HCC cells' capability to proliferate, invade, and migrate while enhancing tumor cell apoptosis. The TCR score could function as a robust and potential tool to predict immune activity and improve clinical outcomes for HCC patients.

RNASET2 Deficiency Induces Hepatocellular Carcinoma Metastasis through Cholesterol-Triggered MET Activation.

Metastasis remains a significant challenge in the treatment of hepatocellular carcinoma (HCC). The role of ribonuclease T2 (RNASET2) in HCC is still uncertain, although it has been reported to have contradictory effects on some cancers. Here, it is demonstrated that RNASET2 knockout leads to significant accumulation of cholesterol, which in turn promotes MET-mediated HCC metastasis. Mechanistically, the absence of RNASET2 hinders the degradation of RNA into uridine, thereby reducing the conversion to UTP. This reduction restrains glucuronate metabolism and the expression of the related enzyme UDP-glucuronosyltransferase (UGT)1A1, ultimately resulting in the accumulation of cholesterol due to decreased formation of glucuronidated-bile acids. The administration of cholesterol induces the migration and invasion of HCC cells through MET (mesenchymal-epithelial transition factor) activation. However, the deficiency of RNASET2-induced HCC metastasis can be reversed by blocking MET with shRNA or savolitinib. The study identifies RNASET2 as a key regulator that coordinates RNA, glucuronate, and cholesterol metabolism. Its deficiency drives HCC metastasis through cholesterol-triggered MET activation. These findings highlight the potential of targeting RNASET2 and MET in improving the prognosis of HCC.

NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma

BackgroundNUAK1 is associated with metastasis and drug resistance in hepatocellular carcinoma (HCC). However, little is known about the immune functions of NUAK1 in HCC. Therefore, the aim of this study was to elucidate the novel role of NUAK1 in facilitating immune evasion in HCC and to investigate the mechanisms underpinning this process.MethodThe levels of NUAK1 expression and the infiltration of CD8+ T cells were assessed in tumor tissues from HCC patients and mice xenograft model. HCC cell lines were used to validate the role of NUAK1 in regulating the transcription of PD-L1, the diethylnitrosamine-induced HCC model was established and the expression levels of NUAK1 and PD-L1 proteins in the rat livers were detected. Western blotting, immunofluorescence, real time PCR, and immunohistochemical staining were used to investigate the underlying mechanisms by which NUAK1 regulates PD-L1 expression in hepatocellular carcinoma.ResultsNUAK1 expression was negatively correlated with CD8+ T cell infiltration in tumor tissues from HCC patients and mice xenograft model. Both gain and loss of functions have identified NUAK1 promoted PD-L1 expression at transcriptional level in HCC cells. The increased expression of NUAK1 and PD-L1 proteins were observed in the rat livers of diethylnitrosamine-induced HCC model. Moreover, overexpression of NUAK1 promotes GSK3β Ser9 phosphorylation, β-catenin expression and nuclear accumulation in HCC cells. By contrast, knockdown of NUAK1 has opposite effects. Inhibition of GSK3β activity significantly promoted β-catenin expression and PD-L1 expression in HCC cells. IHC analyses of tumor tissues from HCC patients suggested that the levels of p-GSK3β and β-catenin were positively correlated with NUAK1 expression. Knockdown of β-catenin also reversed NUAK1-mediated PD-L1 expression in HCC cells.ConclusionsThis study revealed a novel role for NUAK1, which promotes the transcriptional expression of PD-L1 by activating GSK3β/β-catenin signaling pathway, leading to immune escape of hepatocellular carcinoma.Registry and the registration no. of the study/trial: Not applicable.

Single-cell landscape of dynamic changes in CD8+ T cells, CD4+ T cells and exhausted T cells in hepatocellular carcinoma

Hepatocellular carcinoma has a high incidence and poor prognosis. In this study, we investigated the value of T-cell-related genes in prognosis by single-cell sequencing data in hepatocellular carcinoma. Twelve cases of hepatocellular carcinoma single-cell sequencing were included in the study. The high dimensional weighted gene co-expression network analysis (hdWGCNA) was used to identify gene modules associated with CD4+ T cells, CD8+ T cells and exhausted T cells. Altered signaling pathway activity in exhausted T cells was uncovered by the AUCell algorithm. xCELL, TIMER, QUANTISEQ, CIBERSORT and CIBERSORT-abs were performed to explore immune cell infiltration. Immune checkpoint inhibitor genes and TIDE methods were used to predict immunotherapy response. Finally, immunohistochemistry and real-time PCR were used to verify gene expression. The hdWGCNA algorithm identified 40 genes strongly associated with CD4+ T cells, CD8+ T cells and exhausted T cells. Seven genes were finally selected for transcriptome data modeling. The results of the three independent datasets suggested that the model had strong prognostic value. Model genes were critical factors influencing CD4+ T cell and CD8+ T cell infiltration in patients. The efficacy of PD-1 immunotherapy was higher in patients belonging to the low-risk group. Alterations in signaling pathways’ activity within exhausted T cells were crucial factors contributing to the decline in immune function. Differential expression of seven genes in CD8+ T cells, CD4+ T cells and exhausted T cells were key targets for improving immunotherapy response in HCC.

Targeting FDFT1 Reduces Cholesterol and Bile Acid Production and Delays Hepatocellular Carcinoma Progression Through the HNF4A/ALDOB/AKT1 Axis.

Targeting cholesterol metabolism is a novel direction for tumor therapy. Unfortunately, the current use of statins for hepatocellular carcinoma (HCC) is controversial. Herein, farnesyl-diphosphate farnesyltransferase 1 (FDFT1) is identified as a novel target for treating HCC and a potential alternative to statins. Twenty-three key genes in cholesterol biosynthesis are screened, and FDFT1 is identified via public databases (The Cancer Genome Atlas, International Cancer Genome Consortium and Gene Expression Omnibus). Clinical samples reveal that FDFT1 is highly expressed in HCC tissues, and this phenotype is strongly associated with a poor prognosis. Functionally, FDFT1 knockdown inhibits the proliferation and metastasis of HCC cells and suppresses hepatocarcinogenesis in vitro and in vivo, whereas FDFT1 overexpression promotes HCC cell proliferation and metastasis. Mechanistically, FDFT1 downregulation decreases cholesterol and bile acid levels and then increases hepatocyte nuclear factor 4 alpha (HNF4A) transcriptional activity. Experiments indicate that HNF4A combines with the promoter of aldolase B (ALDOB)and promotes the ALDOB transcription and that ALDOB combines with AKT serine/threonine kinase 1 (AKT1) and inhibits AKT1 phosphorylation. Moreover, FDFT1 knockdown combined with AKT inhibitor (AZD5363) treatment shows remarkable therapeutic potential. FDFT1 inhibition reduces cholesterol and bile acid levels to delay HCC progression through the HNF4A/ALDOB/AKT1 axis. Thus, targeting FDFT1 may be a novel potential strategy for treating HCC.

Eltrombopag Inhibited Liver Cancer by Enhancing SMYD4 Protein Degradationvia TRIP12 Ubiquitinase

AbstractAccording to prior studies, SET and MYND domain‐containing protein 4 (SMYD4) is involved in tumor progression and metastasis, representing a potential therapeutic target for tumors. However, no specific inhibitors or drugs targeting SMYD4 are currently available. In this study, molecular docking and molecular dynamics simulations were used to screen small molecule lead compounds binding to SMYD4 protein. CCK8 assay, colony formation assay, EdU assay were used to analyze the viability and proliferation of tumor cells. Flow cytometric analysis was used to evaluate cell apoptosis and cell cycle. Clorazepate, Ativan, Darifenacin and Eltrombopag were found to bind with SMYD4 with the highest probability and to meet the five principles of the drug class. Molecular dynamics simulations showed that Eltrombopag had the strongest binding capacity to SMYD4 protein. The functional analysis showed that Eltrombopag inhibited hepatocellular carcinoma cell proliferation and promoted apoptosis in vivo and in vitro at low density. Moreover, Eltrombopag enhanced ubiquitination of SMYD4 protein and promoted its degradation via thyroid hormone receptor interactor 12(TRIP12). These findings suggest that Eltrombopag is a potential inhibitor of SMYD4 protein, representing a novel leading compound for SMYD4 and applied for tumor treatment.

AFP shields hepatocellular carcinoma from macrophage phagocytosis by regulating HuR-mediated CD47 translocation in cellular membrane.

Alpha fetoprotein(AFP) overexpression connecting with macrophage dysfunction remain poorly defined. In this study, explore AFP regulates macrophage immunomodulation in hepatocellular carcinoma(HCC) through comprehensive in vitro and in vivo studies. Immunohistochemical and immunofluorescence staining was used to analyze the relativity of AFP and cellular membrane CD47 expression in clinical 30 HCC tissues, and the expression of AFP and CD47 in HCC cells. The intelligent living-cell high-throughput imaging analyzer was applied to dynamically track and image of macrophages to phagocytize HCC cells. The effect of AFP on regulating the level of CD47 in cellular membrane and growth of tumor in vivo was performed by animal experiment. The association of AFP and CD47 in HCC cells was detected by single cell analysis. The present results indicated that AFP upregulated the localization of CD47 on the HCC cell surface. CD47 overexpression stimulates HCC to escape immune surveillance by transmitting "don't eat me" signals to macrophages, lead to inhibit macrophage to phagocytize HCC cells. Mechanistically, the results demonstrated that AFP enhanced CD47 membrane translocation by interacting with Hu-Antigen R(HuR), an RNA-binding protein that regulates mRNA stability and translation. AFP alters the subcellular distribution of HuR, increasing its cytoplasmic accumulation and binding to CD47 transcript. AFP enhanced CD47 membrane translocation by interacting with HuR. These findings proved that AFP could inhibit macrophage to phagocytize HCC cells by upregulating the localization of CD47 on the HCC cell surface. Combination of AFP with CD47 blockade may be a potential therapeutic strategy for HCC treatment.

The Oncopig hepatocellular carcinoma model: An innovative large animal platform for evaluating treatment effects.

619 Background: Hepatocellular carcinoma (HCC) is an aggressive disease associated with poor prognosis and limited treatment options. Systemic therapies for advanced HCC include sorafenib, lenvatinib, and atezolizumab plus bevacizumab. However, given the limited efficacy of systemic treatment options, novel treatment approaches including combination of systemic and locoregional therapies (LRTs) is required to improve patient outcomes. These approaches require advanced large animal models to prove effectiveness. Pigs represent an ideal platform that provide both regulatory acceptable and clinically relevant large animal cancer models due to their similar physiology, size, genetics, immunity, and metabolism in relation to humans. The Oncopig HCC model is an ideal tool for testing LRT and other oncolytic based treatments. Methods: The Oncopig cancer model develops tumors following induced expression of KRAS G12D and TP53 R167H driver mutations utilizing an adenoviral vector encoding Cre recombinase (AdCre). Oncopig HCC cell lines were developed by isolating hepatocytes from Oncopig liver biopsies and transformed in vitro via exposure to AdCre. Transgene expression and hepatocyte cell origin were validated by RT-PCR and arginase-1 staining, respectively. Comparison of in vitro Oncopig, human, and murine HCC cell line phenotypes (proliferation, migration, and chemotherapeutic response) was performed. Oncopig HCC tumor formation was performed via autologous intrahepatic injection of Oncopig HCC cells. Tumor formation was evaluated using ultrasound and CT imaging. Results: Oncopig and human HCC cell lines displayed similar cell cycle lengths and migration rates. Oncopig HCC cells were consistently more predictive of human HCC responses than murine cells when treated with standard chemotherapeutic agents (doxorubicin, cisplatin, mitomycin C, and sorafenib). Importantly, consistent with human HCC, Oncopig HCC cells were non-responsive to 5-fluorouracil, while murine HCC cells were responsive. Intrahepatic injection of Oncopig HCC cells resulted in tumors visible on ultrasound and CT scan within 2-4 weeks. Resulting tumors were consistent with human tumors on imaging. Histological analysis of tumor biopsies confirmed the identity of the resulting masses as HCC tumors based on positive arginase-1 and KRAS G12D staining. Conclusions: The Oncopig HCC model may be more predictive of human HCC chemotherapeutic responses than currently employed murine models. Tumors develop rapidly (within 2-4 weeks) and are readily identified on ultrasound and CT imaging, making this an ideal model for evaluation of novel therapeutic approaches. Further exploration and development of additional tumor models including further translational characterization will be important for broader utility.

High mobility group protein N2 inhibits the progression of hepatocellular carcinoma and the related molecular mechanisms.

High mobility group protein N2 (HMGN2) related pathways are involved in chromatin regulation/acetylation. It has been reported to be involved in several types of cancers. A recent sequencing study suggested that HMGN2 might be involved in the progression of hepatocellular carcinoma (HCC). This study aimed to explore the role of HMGN2 in HCC, which has been proven to be involved in the development of HCC. In this study, we collected clinical samples and cultured normal hepatocytes and hepatocellular carcinoma cell lines to detect HMGN2 expression levels using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and western blot assay. Subsequently, to determine the role of HMGN2 in HCC, HMGN2 was overexpressed in HCC cell lines. MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide) assay was used to detect the cell proliferative capacity, and proliferation-related proteins were detected by RT-qPCR and western blot assay. To observe the effect of HMGN2 on cell migration and invasion capacity, Transwell assay was performed. Then, cell apoptosis was detected by flow cytometry, and caspase3 and cleaved-caspase3 were detected using western blot assay. Finally, EMT (epithelial to mesenchymal transition)-related proteins, and matrix metalloproteinase-2 (MMP-2) and MMP-9 expression were detected by RT-qPCR and western blot assay. HMGN2 expression was decreased in HCC tissues as well as in HCC cell lines. After overexpression of HMGN2, MTT results suggested that cell proliferation was decreased, and flow cytometry results showed that the apoptosis level was increased and ki-67 and proliferating cell nuclear antigen (PCNA) expression levels were decreased. On the contrary, cleaved-caspase 3 expression level was increased. HCC cells overexpressing HMGN2 showed a drastic reduction in the number of migrating and invading cells, and the expression levels of MMP-2 and MMP-9 were significantly decreased. Finally, E-cadherin expression was elevated in HCC cells transfected with the HMGN2-plasmid, while N-cadherin showed the opposite result. HMGN2 expression was significantly decreased in patients with HCC. HMGN2 inhibits the malignant behavior of HCC cells and is a potential therapeutic target for HCC.

Mitochondrial damage and associated combined toxicity induced by deoxynivalenol and Alternaria toxins co-exposure

Deoxynivalenol and Alternaria toxins are common food contaminants posing significant threats to human and animal health. Although their individual toxicities have been extensively studied, the combined effects of co-exposure remain largely unexplored. Our findings revealed combined toxins displayed concentration-dependent synergistic and antagonistic interactions on human hepatocellular carcinoma cells. Co-exposure significantly reduced mitochondrial activity, increased intracellular ROS levels, and activated the mitochondrial-dependent caspase signaling pathway, ultimately leading to enhanced apoptosis. Metabolomics analysis revealed that combined exposure severely disrupted multiple key metabolic processes, including those related to energy (NAD+, adenosine, AMP, and ADP), amino acids (l-aspartate, L-glutamate), and carbohydrate (Glucose-6-phosphate) metabolisms. These findings highlight that co-occurrence exacerbates the disruption of overall metabolic balance, potentially impairing the normal function of cells by affecting energy production, protein synthesis, and cell proliferation. This study underscores the importance of considering combined mycotoxin exposure and provided possible ideas for inhibiting or mitigating toxicity to ensure food safety.

Therapeutic Effects of Crocin Nanoparticles Alone or in Combination with Doxorubicin against Hepatocellular Carcinoma In vitro.

Crocin (CRO), the primary antioxidant in saffron, is known for its anticancer properties. However, its effectiveness in topical therapy is limited due to low bioavailability, poor absorption, and low physicochemical stability. This study aimed to prepare crocin nanoparticles (CRO-NPs) to enhance their pharmaceutical efficacy and evaluate the synergistic effects of Cro-NPs with doxorubicin (DOX) chemotherapy on two cell lines: human hepatocellular carcinoma cells (HepG2) and non-cancerous cells (WI38). CRO-NPs were prepared using the emulsion diffusion technique and characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), Zeta potential, and Fourier transform infrared spectroscopy (FT-IR). Cell proliferation inhibition was assessed using the MTT assay for DOX, CRO, CRO-NPs, and DOX+CRO-NPs. Apoptosis and cell cycle were evaluated by flow cytometry, and changes in the expression of apoptotic gene (P53) and autophagic genes (ATG5 & LC3) were analyzed using real-time polymerase chain reaction. TEM and SEM revealed that CRO-NPs exhibited a relatively spherical shape with an average size of 9.3 nm, and zeta potential analysis indicated better stability of CRO-NPs compared to native CRO. Significantly higher antitumor effects of CRO-NPs were observed against HepG2 cells (IC50 = 1.1 mg/ml and 0.57 mg/ml) compared to native CRO (IC50 = 6.1 mg/ml and 3.2 mg/ml) after 24 and 48 hours, respectively. Annexin-V assay on HepG2 cells indicated increased apoptotic rates across all treatments, with the highest percentage observed in CRO-NPs, accompanied by cell cycle arrest at the G2/M phase. Furthermore, gene expression analysis showed upregulation of P53, ATG5, and LC3 genes in DOX/CRO-NPs co-treatment compared to individual treatments. In contrast, WI38 cells exhibited greater sensitivity to DOX toxicity but showed no adverse response to CRONPs. Although more in vivo studies in animal models are required to corroborate these results, our findings suggest that CRO-NPs can be a potential new anticancer agent for hepatocellular carcinoma. Moreover, they have a synergistic effect with DOX against HepG2 cells and mitigate the toxicity of DOX on normal WI38 cells.

CNDP1 Suppresses the Malignant Behavior of Hepatoma Cell via Restricting PI3K-AKT-mTOR Activation.

Hepatocellular carcinoma (HCC) is a global health problem with increasing morbidity and mortality, and exploring the diagnosis and treatment of HCC at the gene level has been a research hotspot in recent years. In this paper, a series of differentially expressed genes were found from the biochip related to HCC by bioinformatic analysis, then CNDP1 was finally selected for in-depth study according to the function and research progress of each gene. As the rate-limiting enzyme of carnosine hydrolysis, CNDP1 participates in the progress of many diseases, but its function has not been revealed in HCC. In the follow-up study, the low expression of CNDP1 in liver cancer tissues and cells was verified, then the pcDNA3.1-CNDP1 was used to improve the expression level of CNDP1 in HCC cell lines. Furthermore, this paper found that CNDP1 overexpression could significantly suppress cell prolifer-ation, migration, and invasion of HCC cell lines. Mechanismly, the GeneMANIA database predicted that CNDP1 could interact with various proteins that regulate the PI3K-AKT-mTOR signaling pathway, which is overactivated in HCC. And this study showed that CNDP1 overexpression could effectively inhibit the activation of PI3K-AKT-mTOR signaling pathways, more significantly, inhibition of PI3K-AKT-mTOR signaling pathway could disrupt the anti-cancer effect of CNDP1 on HCC. In conclusion, we confirmed that CNDP1 was lowly expressed in HCC tissues and cells, and had potential anti-cancer activity. This discovery will lay a cytological foundation for expanding the biological function of CNDP1 and the diagnosis and treatment of HCC in the future.