Huh7 Cells Research Articles

Synthesis and cytotoxic activity of madecassic acid-silybin conjugate compounds in liver cancer cells.

A series of 14 conjugates of 2α,3β,23-triacetyl-madecassic acid and silybin were designed and synthesized. The madecassic acid unit was linked to silybin either directly at position C-7 or C-3; or through an amino acid linker (glycine, β-alanine, or 11-aminoundecanoic acid) at position C-3. The conjugates were tested in vitro for their cytotoxic effect on HepG2 cells using the MTT assay. The results confirmed that the conjugated compounds demonstrated a stronger cytotoxic effect compared to the parent compounds. Of these compounds, the most promising conjugate, compound 8, was evaluated for cytotoxic activity in the additional Hep3B, Huh7, and Huh7R human hepatocellular carcinoma cell lines and also for cell cycle changes and induction of apoptosis in HepG2 cells. This compound caused a rapid and significant induction of caspase 3 activity and induced cell cycle arrest in the S phase - effects distinct from the activity of madecassic acid. This is the first study on the synthesis and cytotoxicity of madecassic acid-silybin conjugates, and of their testing against liver cancer cell lines and provides evidence for a distinct biological profile versus madecassic acid alone.

Interferon-stimulated gene (ISG12a) suppresses hepatitis B virus replication in Huh 7 cells line

Hepatitis B virus (HBV) and its associated chronic liver disease pose a significant health hazard. Chronic HBV infection is a major contributor to the development of liver cirrhosis, liver fibrosis, and liver cancer. This study focused on ISG12a and its inhibitory effect in Huh7 cells on HBV gene expression and replication. The mammalian hepatoma cells Huh7 were transfected with the pHBV1.3 vector (pCAGGS) or HA-tagged ISG12a to determine the overexpression, to test this hypothesis further, we did knockdown or silencing by transducing Huh7 cells with lentiviruses containing shRNAs targeting ISG12a or scramble control shRNA (shControl). The expression of ISG12a was evaluated through western blot analysis. The HBsAg and HBeAg secretion in the Huh7 cells’ culture media was examined using an ELISA test. The qRT-PCR confirmed the mRNA and 3.5 mRNA Kb of ISG12a. The HBV total RNA was extracted and evaluated through a Northern blot. The isolated DNA was detected through qPCR. Additionally, a mechanistic study of enhancer II (EnhII/Cp) was examined through luciferase reporter testing. Our research findings indicate that ISG12a, an interferon-stimulated gene (ISG), plays a crucial role in suppressing the replication and gene expression of HBV. According to our study using the Huh7 cell system, overexpression of ISG12a resulted in a notable reduction in HBV protein levels as well as intracellular core-associated DNA and RNA levels. On the other hand, silencing ISG12a in Huh7 cells resulted in increased HBV RNA transcripts, DNA, and secreted proteins, indicating that ISG12a plays a role in suppressing HBV replication. Additionally, the research revealed that ISG12a inhibits the function of the EnhII/Cp promoter, which results in reduced HBV gene expression. The EnhII/Cp promoter is involved in regulating HBV gene expression, and the study showed that ISG12a restricts its activity. ISG12a may have a regulatory role in controlling the expression of HBV genes. These findings highlight the importance of ISG12a in HBV gene expression and provide valuable insights for understanding its antiviral function.

PGV-1 Causes Disarrangement of Spindle Microtubule Organization Resulting in Aberrant Mitosis in HLF and HuH6 Cells Associated with Altered MYCN Status.

The HLF and HuH-6 cell lines represent hepatocellular carcinoma (HCC) with different characteristics in chromosome content that may give different drug responses. Here, PGV-1 was intended to challenge the growth-suppressing effect on HLF and HuH-6 and trace the molecular target mechanism of action compared to sorafenib. We applied MTT cytotoxic assay, colony forming assay, flow cytometry analysis, immunofluorescence assay and western blot assay. PGV-1 exhibited cytotoxic effects on HLF and HuH-6 with IC-50 values of 1 µM and 2 µM, respectively, whereas sorafenib showed less cytotoxicity with IC-50 values of 5 µM and 8 µM respectively. PGV-1 suppressed the cell growth permanently but not for sorafenib. Sorafenib did not change the cell cycle profiles on both cells, but PGV-1 arrested the cells at G2/M with the characteristic of senescent cells and mitotic disarrangement. PGV-1 and sorafenib showed the same effect in downregulating p-EGFR, indicating that both compounds have the same target on EGFR activation or as Tyrosine kinase inhibitors. PGV-1 suppressed the MYCN expression in HuH-6 and HLF cells but stabilized cMYC-T58 indicating that even though the MYCN was downregulated, the cells maintained the active form of cMYC. In this regard, PGV-1 also stabilized the expression of PLK-1 and AurA. PGV-1 elicits stronger cytotoxic properties compared to sorafenib. The lower the MYCN expression, the higher the cytotoxic effect of PGV-1. PGV-1 abrogates cell cycle progression of both cells in mitosis through EGFR inhibition and stabilizes PLK-1 and AurA in correlation with the suppression of MYCN expression.

NCAPD2 augments the tumorigenesis and progression of human liver cancer via the PI3K‑Akt‑mTOR signaling pathway.

Non‑SMC condensin I complex subunit D2 (NCAPD2) is a newly identified oncogene; however, the specific biological function and molecular mechanism of NCAPD2 in liver cancer progression remain unknown. In the present study, the aberrant expression of NCAPD2 in liver cancer was investigated using public tumor databases, including TNMplot, The Cancer Genome Atlas and the International Cancer Genome Consortium based on bioinformatics analyses, and it was validated using a clinical cohort. It was revealed that NCAPD2 was significantly upregulated in liver cancer tissues compared with in control liver tissues, and NCAPD2 served as an independent prognostic factor and predicted poor prognosis in liver cancer. In addition, the expression of NCAPD2 was positively correlated with the percentage of Ki67+ cells. Finally, single‑cell sequencing data, gene‑set enrichment analyses and in vitro investigations, including cell proliferation assay, Transwell assay, wound healing assay, cell cycle experiments, cell apoptosis assay and western blotting, were carried out in human liver cancer cell lines to assess the biological mechanisms of NCAPD2 in patients with liver cancer. The results revealed that the upregulation of NCAPD2 enhanced tumor cell proliferation, invasion and cell cycle progression at the G2/M‑phase transition, and inhibited apoptosis in liver cancer cells. Furthermore, NCAPD2 overexpression was closely associated with the phosphatidylinositol 3‑kinase (PI3K)‑Akt‑mammalian target of rapamycin (mTOR)/c‑Myc signaling pathway and epithelial‑mesenchymal transition (EMT) progression in HepG2 and Huh7 cells. In addition, upregulated NCAPD2 was shown to have adverse effects on overall survival and disease‑specific survival in liver cancer. In conclusion, the overexpression of NCAPD2 was shown to lead to cell cycle progression at the G2/M‑phase transition, activation of the PI3K‑Akt‑mTOR/c‑Myc signaling pathway and EMT progression in human liver cancer cells.

Induction of Apoptosis and ROS Production in Liver Cancer Cells by Saponin Fraction from Alcea rosea L. Seeds

Background: Natural compounds are essential targets in anticancer research due to the toxic effects of current chemotherapy and drug resistance. Methods: Saponin extraction from Alcea rosea L. was carried out using 20% methanol and liquid-liquid extraction coupled with the Solid phase extraction (SPE) approach. The saponin fraction was explored for its cytotoxic activity against Huh-7 and MDA-MB-231 cancer cell lines and a non–cancer cell line (HUVEC). The cytotoxic activity and oxidative stress were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2,7 dichlorofluorescein diacetate dyes respectively. The apoptotic potential was investigated using DAPI and acridine orange-ethidium bromide dual staining. Result: The saponin fraction exhibited cytotoxic potential against the cell lines investigated. The IC50 values of the saponin fraction were 49.02, 300 and 325 µg\mL against Huh-7, MDA-MB-231 and HUVEC cell lines, respectively. The cytotoxicity for the extract was dose-dependent. The saponin fraction calculated selectivity index (SI) value indicated high selectivity towards Huh-7 cells (SI = 6.62) compared with normal HUVEC cells. Huh-7 cells treated with saponin fraction showed cellular and nuclear morphological changes, such as apoptotic body formation and chromatin condensation, as observed using light and fluorescent microscopy. However, further investigation is required to assess the the saponin fraction as a potential therapeutic agent.

Glioma-associated oncogene (GLI)-specific decoy oligodeoxynucleotide induces apoptosis and attenuates proliferation, colony formation, and migration in liver cancer cells

Aim: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated death. The Sonic Hedgehog (SHH) signaling pathway participates in the initiation, progression, migration, and recurrence of HCC cancer stem cells. Furthermore, SHH regulates various cellular behaviors such as proliferation, differentiation, survival, self-renewal, epithelial-mesenchymal transition (EMT), and SHH autoregulation. Glioma-associated oncogene (GLI) family zinc finger are key transcription factors in the development of many organs and are deregulated in cancer. In this study, Huh-7 cells were treated with GLI-specific decoy oligodeoxynucleotide (ODN) to evaluate its anticancer impact. Methods: The transfection efficiency of GLI-specific decoy ODN was measured using fluorescent microscopy. Then, the effects of GLI-specific decoy ODN on apoptosis, viability, proliferation rate, colony formation, and migration capacities of Huh-7 cells were assessed. Furthermore, the expression of genes associated with the alteration of SHH was assessed. Results: Treatment of Huh-7 cells with GLI-specific decoy ODN decreased cell viability (56.36% ± 3%). Expression of certain genes such as c-MYC , SNAI2 , ZEB1 , and PROM1 decreased dramatically, while the expression of CDH1 increased significantly. Furthermore, the treated cells’ proliferation, colony formation, and migration capacity decreased considerably. This treatment induced apoptosis in the Huh-7 cells. Conclusion: Inhibition of the SHH signaling pathway using GLI-specific decoy ODN led to a decline in the growth rate of HCC cells, decreased migration, and attenuated EMT progression.

Pharmacological induction of the hypoxia response pathway in Huh7 hepatoma cells limits proliferation but increases resilience under metabolic stress

The hypoxia response pathway enables adaptation to oxygen deprivation. It is mediated by hypoxia-inducible factors (HIF), which promote metabolic reprogramming, erythropoiesis, angiogenesis and tissue remodeling. This led to the successful development of HIF-inducing drugs for treating anemia and some of these molecules are now in clinic. However, elevated levels of HIFs are frequently associated with tumor growth, poor prognosis, and drug resistance in various cancers, including hepatocellular carcinoma (HCC). Consequently, there are concerns regarding the recommendation of HIF-inducing drugs in certain clinical situations. Here, we analyzed the effects of two HIF-inducing drugs, Molidustat and Roxadustat, in the well-characterized HCC cell line Huh7. These drugs increased HIF-1α and HIF-2α protein levels which both participate in inducing hypoxia response genes such as BNIP3, SERPINE1, LDHA or EPO. Combined transcriptomics, proteomics and metabolomics showed that Molidustat increased the expression of glycolytic enzymes, while the mitochondrial network was fragmented and cellular respiration decreased. This metabolic remodeling was associated with a reduced proliferation and a lower demand for pyrimidine supply, but an increased ability of cells to convert pyruvate to lactate. This was accompanied by a higher resistance to the inhibition of mitochondrial respiration by antimycin A, a phenotype confirmed in Roxadustat-treated Huh7 cells and Molidustat-treated hepatoblastoma cells (Huh6 and HepG2). Overall, this study shows that HIF-inducing drugs increase the metabolic resilience of liver cancer cells to metabolic stressors, arguing for careful monitoring of patients treated with HIF-inducing drugs, especially when they are at risk of liver cancer.

Clinacanthus nutans (Burm. f.) Lindau Extract Inhibits Dengue Virus Infection and Inflammation in the Huh7 Hepatoma Cell Line.

Dengue virus (DENV) infection has emerged as a global health problem, with no specific treatment available presently. Clinacanthus nutans (Burm. f.) Lindau extract has been used in traditional medicine for its anti-inflammatory and antiviral properties. We thus hypothesized C. nutans had a broad-ranged activity to inhibit DENV and the liver inflammation caused by DENV infection. The study showed that treatment using C. nutans extract during DENV infection (co-infection step) showed the highest efficiency in lowering the viral antigen concentration to 22.87 ± 6.49% at 31.25 μg/mL. In addition, the virus-host cell binding assay demonstrated that C. nutans treatment greatly inhibited the virus after its binding to Huh7 cells. Moreover, it could remarkably lower the expression of cytokine and chemokine genes, including TNF-α, CXCL10, IL-6, and IL-8, in addition to inflammatory mediator COX-2 genes. Interestingly, the activation of the NF-κB signaling cascade after C. nutans extract treatment was dramatically decreased, which could be the underlying mechanism of its anti-inflammatory activity. The HPLC profile showed that gallic acid was the bioactive compound of C. nutans extract and might be responsible for the antiviral properties of C. nutans. Taken together, our results revealed the potential of C. nutans extract to inhibit DENV infection and lower inflammation in infected cells.

Bioactive alkaloids from Nepalese Corydalis chaerophylla D.C. acting on the regulation of PCSK9 and LDL-R in vitro.

Four new alkaloids Chaeronepaline-A (1), Chaeronepaline-B (2), Chaeronepaline-C (3), and Chaeronepaline-D (4) were isolated from Corydalis chaerophylla D.C. collected from Nepal and their structures were elucidated by spectroscopic data, 1D, 2D NMR and mass spectrometry. The structures were established as 3,12- Dimethoxy-5,6-dihydroisoquinolino [2,1-b] isoquinolin- 7- ium- 2, 9- diol (1), 7-methyl-5, 6, 7, 8-tetrahydroisoquinoline- 2, 3- methylenedioxy- (8-> 9)- 10, 12- methylenedioxy- benzoic-16-acid (2), 7- methyl-5, 6, 7, 8- tetrahydro- 8H-spiro-9,14-dihydroxy-11,12-methylenedioxy-indane-isoquinoline (3) and 7- methyl-5, 6, 7, 8- tetrahydro- 8H-spiro-9,14-dihydroxy-11,12-methylenedioxy-indane-isoquinoline-N-oxide (4). The new alkaloids were tested in human hepatoma cell line to assess their ability to modulate the expression of low-density lipoprotein receptor (LDL-R), of proprotein convertase subtilisin/kexin 9 (PCSK9) and to affect cellular cholesterol biosynthesis with the aim to evaluate their potential hypocholesterolemic effect. Results indicated that compounds 2 and 3 upregulate the LDLR, and inhibited the cholesterol biosynthesis with compound 2, which also reduced the secretion of PCSK9 by Huh7 cells. These in vitro data indicated a potential hypocholesterolemic effect of compound 2 that requires further in vivo validation.

Exploring the anti-hepatocellular carcinoma effects of Xianglian Pill: Integrating network pharmacology and RNA sequencing via in silico and in vitro studies

BackgroundLiver cancer represents a most common and fatal cancer worldwide. Xianglian Pill (XLP) is an herbal formula holding great promise in clearing heat for treating diseases in an integrative and holistic way. However, due to the complex constituents and multiple targets, the exact molecular mechanisms of action of XLP are still unclear. PurposeThis study is focused on hepatocellular carcinoma (HCC), the most common type of liver cancer. The aim of this study is to develop a fast and efficient model to investigate the anti-HCC effects of XLP, and its underlying mechanisms. Materials and methodsHepG2, Hep3B, Mahlavu, HuH-7, or Li-7 cells were employed in the studies. The ingredients were analyzed using liquid chromatography tandem mass spectrometry (LC-MS). RNA sequencing combined with network pharmacology was used to elucidate the therapeutic mechanism of XLP in HCC via in silico and in vitro studies. An approach was constructed to improve the accuracy of prediction in network pharmacology by combining big data and omics. ResultsFirst, we identified 13 potential ingredients in the serum of XLP-administered rats using LC-MS. Then the network pharmacology was performed to predict that XLP demonstrates anti-HCC effects via targeting 94 genes involving in 13 components. Modifying the database thresholds might impact the accuracy of network pharmacology analysis based on RNA sequencing data. For instance, when the matching rate peak is 0.43, the correctness rate peak is 0.85. Moreover, 9 components of XLP and 6 relevant genes have been verified with CCK-8 and RT-qPCR assay, respectively. ConclusionBased on the crossing studies of RNA sequencing and network pharmacology, XLP was found to improve HCC through multiple targets and pathways. Additionally, the study provides a way to optimize network pharmacology analysis in herbal medicine research.

Metal-Organic Framework: Fabrication of Nano Fluorescent Composite Materials and Treatment of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a common malignant tumor originating from liver cells, characterized by complex pathogenesis and limited treatment options such as surgery, chemotherapy, and transplantation. Cisplatin, an effective chemotherapeutic agent, disrupts cancer cell DNA but is hindered by side effects and the need for controlled sustained release to optimize efficacy. Metal-organic frameworks (MOFs) have emerged as promising nanocarriers for precise local drug delivery, reducing required doses and mitigating side effects of chemotherapeutic drugs, thus offering a potential avenue for hepatocellular carcinoma (HCC) treatment. In this research, a rectangular channel MOF (Rumgay H, Ferlay J, Martel C, Georges D, Ibrahim AS, Zheng R, Wei W, Lemmens VEPP, Soerjomataram I (2022) Global, regional and national burden of primary liver cancer by subtype. Eur J Cancer 161:108-118) carrier was synthesized using ligand L as the organic linker coordinated with Cu(II) and I(I). The MOF's structure and fluorescence properties were characterized. Additionally, to enhance substrate biocompatibility, composite carrier materials were prepared by incorporating polylactic acid (PLA) with 1, utilized for cisplatin loading. To evaluate the inhibitory effect of PLA-1@cisplatin on HCC, HepG-2 and Huh-7 HCC cell lines were treated with varying concentrations of the drug for 48h, and their cell viability was assessed. The results demonstrated a significant dose-dependent reduction in cell viability of both HepG-2 and Huh-7 cells. To explore the potential inhibitory mechanism of PLA-1@cisplatin on HCC, the mRNA levels of GADD45A and NACC1 in HepG-2 and Huh-7 cells post-treatment were measured. GADD45A expression, initially low in HCC cells, was significantly upregulated after drug treatment, while NACC1, typically highly expressed in HCC, showed a significant decrease in mRNA levels with increasing concentrations of PLA-1@cisplatin. These findings indicate that PLA-1@cisplatin effectively upregulates GADD45A expression and downregulates NACC1 expression. Overall, the developed cisplatin-loaded nanoparticle system holds promise for HCC treatment by reducing chemotherapy side effects and enhancing drug efficacy.

Curzerene suppresses hepatocellular carcinoma progression through the PI3K/AKT/MTOR pathway.

Hepatocellular carcinoma (HCC) is one of the most aggressive cancers worldwide. Curzerene is a sesquiterpene and component of Curcuma rhizomes and has anti-tumor and anti-inflammatory properties. The study aimed to investigate the effects of curzerene on the malignant phenotypes and tumor growth in HCC. Various concentrations of curzerene were used to treat human HCC cells (Huh7 and HCCLM3). Cell viability, apoptosis, cell cycle, invasion, and migration were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, Transwell, and wound healing assays. Cell cycle-, apoptosis-, and signaling pathway-related proteins were analyzed by Western blot analysis. A mouse xenograft model was established to analyze the anti-tumor effects of curzerene in vivo. Curzerene repressed the proliferation, invasion, and migration of Huh7 and HCCLM3 cells. Curzerene also induced G2/M cycle arrest and cell apoptosis. Curzerene downregulated the CDK1, cyclin B1, PCNA, Bcl-2, matrix metallopeptidases (MMP)2, and MMP9 protein expression and upregulated the Bax, cleaved caspase3, and cleaved poly ADPribose polymerase protein expression in HCC cells. Curzerene restrained the phosphorylation of PI3K, AKT, and the Mammalian target of rapamycin (mTOR) in Huh7 and HCCLM3 cells. The in vivo data revealed that curzerene inhibited HCC tumor growth and decreased the expression of phosphorylated mTOR in xenograft mouse models. Curzerene inhibited cell malignancy in vitro and tumor growth in vivo in HCC, suggesting that curzerene may be a candidate agent for anti-HCC therapy.

Ticlopidine protects hepatic ischemia-reperfusion injury via suppressing ferroptosis

Hepatic ischemia-reperfusion injury (IRI) is a major cause of liver damage during hepatic resection, transplantation, and other surgical procedures, often leading to graft failure and liver dysfunction. Recent studies have identified ferroptosis, a form of regulated cell death characterized by iron-dependent lipid peroxidation, as a key contributor to IRI. In this study, we investigated the protective effects of Ticlopidine, a thienopyridine compound and platelet aggregation inhibitor, on hepatic IRI. Using a C57BL/6J mouse model, we demonstrated that prophylactic Ticlopidine treatment significantly reduced necrotic and fibrotic areas in liver tissues, as well as serum levels of alanine transaminase (ALT) and aspartate aminotransferase (AST). Prussian Blue staining revealed that Ticlopidine pretreatment decreased iron accumulation in hepatic tissues, whereas markers of lipid peroxidation (malondialdehyde and 4-hydroxynonenal) and ferroptosis (PTGS2) were significantly downregulated. Additionally, Ticlopidine ameliorated inflammatory infiltration as indicated by reduced Gr-1 staining. In vitro, Ticlopidine dose-dependently inhibited ferroptosis induced by various inducers in liver cancer cell lines HUH7 and fibrosarcoma cells HT1080. The protective effects involved partial rescue of lipid peroxidation, significant reduction of ferrous iron levels, and strong protection against mitochondrial damage. These findings suggested that Ticlopidine acts as a broad-spectrum ferroptosis inhibitor, offering a promising therapeutic approach for protecting the liver against IRI.

ELOVL1 is upregulated and promotes tumor growth in hepatocellular carcinoma through regulating PI3K-AKT-mTOR signaling

BackgroundThe functions of the ELOVLs are mainly involved in the elongation of saturated and polyunsaturated fatty acids, thus influencing the metabolism of fatty acids. Abnormal lipid metabolism may result in NAFLD and NASH, which may lead to cirrhosis and liver cancer. These results suggest that ELOVLs-mediated metabolism might be involved in the development of HCC. The purpose of this study was to study the expression and function of ELOVL1 in human liver cancer. MethodUsing TCGA, GEPIA and other databases, we analyzed the relationship between the expression of ELOVL1 and liver cancer. The expression of ELOVL1 was detected by immunohistochemical method and Western blot method in hepatic carcinoma and hepatic carcinoma cells. Then, the effects of ELOVL1 on proliferation, apoptosis and invasion in vitro and in vivo were investigated by means of different methods. ResultOur results indicate that ELOVL1 is more highly expressed in liver cancer than in normal tissues. Survival analysis showed that OS and DSS were shorter in patients with high ELOVL1 expression than in those with low expression. Multivariate Cox analysis further demonstrated that over-expression of ELOVL1 was an independent risk factor for overall survival in HCC. The results of ROC also confirmed the value of ELOVL1 in the diagnosis of liver cancer. The results of KEGG enrichment and GSEA indicate that ELOVL1 is associated with lipid metabolism and NAFLD, as well as PPAR, PI3K-AKT-mTOR. Compared with the control group, it was found that silencing ELOVL1 in Huh7 and HepG2 cells could inhibit the growth of cells, promote the apoptosis and decrease the metastasis and invasion. Changes in ELOVL1 induced cell proliferation and metastasis may be related to PI3K/AKT/mTOR. Low expression of ELOVL1 inhibited the growth of xenograft tumors in hepatocellular carcinoma xenograft model. ConclusionOur data indicate that the activation of PI3K/AKT/mTOR pathway in HCC may contribute to the promotion of cancer. Thus, ELOVL1 may be a promising therapeutic target for HCC.

Targeted Isolation of Antiviral Labdane Diterpenes from the Bark of Neo-uvaria foetida (Annonaceae) using LC-MS/MS-Based Molecular Networking.

In the search of new inhibitors for human coronavirus (HCoV), we screened extracts of endemic Annonaceae plants on an assay using a cellular model of Huh-7 cells infected with the human alphacoronavirus HCoV-229E. The EtOAc bark extract of the rare Southeast Asian plant Neo-uvaria foetida exhibited inhibition of HCoV-229E and SARS-CoV-2 viruses with IC50 values of 3.8 and 7.8 μg/mL, respectively. Using LC-MS/MS and molecular networking analysis guided isolation, we discovered two new labdane-type diterpenoids, 8-epi-acuminolide (1) and foetidalabdane A (4), and three known labdane diterpenoids, acuminolide (2), 17-O-acetylacuminolide (3), and spiroacuminolide (5). A new norlabdane diterpene, 16-foetinorlabdoic acid (6), was also isolated and identified. Excluding compounds 5 and 6, all other metabolites were active against the virus HCoV-229E. Terpenoids 1 and 4 presented antiviral activity against SARS-CoV-2 with IC50 values of 63.3 and 93.5 μM, respectively, indicating lower potency. Additionally, virological assays demonstrated that compounds 1, 2, and 3 exert antiviral effects against Zika virus by specifically interfering with the late stage of its infectious cycle with IC50 values of 76.0, 31.9, and 14.9 μM, respectively.

Analysis of the susceptibility of refractory hepatitis C virus resistant to nonstructural 5A inhibitors

Resistance-associated substitutions (RASs) of hepatitis C virus (HCV) affect the efficacy of direct-acting antivirals (DAAs). In this study, we aimed to clarify the susceptibility of the coexistence of nonstructural (NS) 5A Q24K/L28M/R30Q (or R30E)/A92K RASs, which were observed in patients with DAAs re-treatment failure and to consider new therapeutic agents. We used a subgenomic replicon system in which HCV genotype 1B strain 1B-4 was electroporated into OR6c cells derived from HuH-7 cells (Wild-type [WT]). We converted WT genes to NS5A Q24K/L28M/R30Q/A92K or Q24/L28K/R30E/A92K. Compared with the WT, the Q24K/L28M/R30Q/A92K RASs was 36,000-fold resistant to daclatasvir, 440,000-fold resistant to ledipasvir, 6300-fold resistant to velpatasvir, 3100-fold resistant to elbasvir, and 1.8-fold resistant to pibrentasvir. Compared with the WT, the Q24K/L28M/R30E/A92K RASs was 640,000-fold resistant to daclatasvir and ledipasvir, 150,000-fold resistant to velpatasvir, 44,000-fold resistant to elbasvir, and 1500-fold resistant to pibrentasvir. The Q24K/L28M/R30E/A92K RASs was 816.3 times more resistant to pibrentasvir than the Q24K/L28M/R30Q/A92K RASs. Furthermore, a combination of pibrentasvir and sofosbuvir showed therapeutic efficacy against these RASs. Combination regimens may eradicate HCV with NS5A Q24K/L28M/R30E/A92K RASs.

Superior cuproptotic efficacy of diethyldithiocarbamate-Cu4O3 nanoparticles over diethyldithiocarbamate-Cu2O nanoparticles in metastatic hepatocellular carcinoma.

Metastatic hepatocellular carcinoma (HC) is a serious health concern. The stemness of cancer stem cells (CSCs) is a key driver for HC tumorigenesis, apoptotic resistance, and metastasis, and functional mitochondria are critical for its maintenance. Cuproptosis is Cu-dependent non-apoptotic pathway (mitochondrial dysfunction) via inactivating mitochondrial enzymes (pyruvate dehydrogenase "PDH" and succinate dehydrogenase "SDH"). To effectively treat metastatic HC, it is necessary to induce selective cuproptosis (for halting cancer stemness genes) with selective oxidative imbalance (for increasing cell susceptibility to cuproptosis and inducing non-CSCs death). Herein, two types of Cu oxide nanoparticles (Cu4O3 "C(I + II)" NPs and Cu2O "C(I)" NPs) were used in combination with diethyldithiocarbamate (DD, an aldehyde dehydrogenase "ALDH" inhibitor) for comparative anti-HC investigation. DC(I + II) NPs exhibited higher cytotoxicity, mitochondrial membrane potential, and anti-migration impact than DC(I) NPs in the treated human HC cells (HepG2 and/or Huh7). Moreover, DC(I + II) NPs were more effective than DC(I) NPs in the treatment of HC mouse groups. This was mediated via higher selective accumulation of DC(I + II) NPs in only tumor tissues and oxidant activity, causing stronger selective inhibition of mitochondrial enzymes (PDH, SDH, and ALDH2) than DC(I)NPs. This effect resulted in more suppression of tumor and metastasis markers as well as stemness gene expressions in DC(I + II) NPs-treated HC mice. In addition, both nanocomplexes normalized liver function and hematological parameters. The computational analysis found that DC(I + II) showed higher binding affinity to most of the tested enzymes. Accordingly, DC(I + II) NPs represent a highly effective therapeutic formulation compared to DC(I) NPs for metastatic HC.

The 3,3'-dimethoxy-4,4'-dihydroxy-stilbene Triazole (STT) Inhibits Liver Cancer Cell Growth by Targeting Akt/mTOR Pathway.

The present study was aimed to investigate the proliferation inhibitory ability of 3,3'-dimethoxy-4,4'-dihydroxy-stilbene triazole (STT) on SNU449 and Huh7 cells. Moreover, the mechanism associated with the suppression of liver cancer cell proliferation by STT was also studied. The results revealed that STT suppresses proliferation of SNU449 and Huh7 cells to 28 and 21%, respectively treatment with 20 µM. The clonogenic survival of SNU449 and Huh7 cells was also significantly reduced after incubation with STT compared to the control cultures. In comparison to the control, STT treatment significantly decreased the invasive potential of SNU449 cells. Treatment with STT led to a prominent suppression in p62 and increase in LC3B protein expression in SNU449 cells compared to the control cells. The STT treatment dramatically decreased p-Akt and p-mTOR protein expression in SNU449 cells. Docking study revealed that STT interacts via traditional hydrogen bonding with the glutamine, phenylalanine, leucine, serine, arginine, aspartic acid, and lysine residues of Akt protein. In summary, the current study demonstrates that STT effectively suppresses the viability of SNU449 and Huh7 liver cancer cells. Moreover, STT treatment of the liver cancer cells also significantly reduces the clonogenic survival and invasive potential of SNU449 cells. Treatment of liver cancer cells with STT increases the expression of autophagic, targets anti-autophagic protein expression and down-regulates Akt/mTOR pathway to inhibit cancer growth and proliferation. Thus, STT exhibits prominent anticancer effect and needs to be investigated further as a potential candidate for the treatment of liver cancer.

TGF-β1-Induced LINC01094 promotes epithelial-mesenchymal transition in hepatocellular carcinoma through the miR-122-5p/TGFBR2-SAMD2-SMAD3 Axis.

Hepatocellular carcinoma (HCC) is a common malignancy with a poor prognosis. It has been proven that long non-coding RNAs (lncRNAs) play an essential role in regulating HCC progression. However, the involvement of LINC01094 in regulating epithelial-mesenchymal transition (EMT) in HCC remains unclear. LINC01094 expression in HCC patients was retrieved from the Cancer Genome Atlas database. Overexpressing and downregulating LINC01094 were conducted to investigate its biological functions using Hep3B, SNU-387, and HuH-7 cells. Western blotting and morphological observation were performed to study the EMT in HCC cells. Transwell assay was adopted to determine the migration and invasion of HCC cells. The underlying mechanism of competitive endogenous RNAs (ceRNAs) was investigated using bioinformatics analysis, quantitative reverse-transcription polymerase chain reaction, and rescue experiments. Elevated LINC01094 expression was observed in HCC and associated with a poor prognosis. Knockdown of LINC01094 expression in SNU-387 and HuH-7 cells could inhibit migration, invasion, and EMT markers. Overexpression of LINC01094 indicated that LINC01094 promoted EMT via the TGF-β/SMAD signaling pathway. The bioinformatics analysis revealed that miR-122-5p was a target of LINC01094. The miRWalk database analysis showed that TGFBR2, SMAD2, and SMAD3 were downstream targets of miR-122-5p. Mechanically, LINC01094 acted as a ceRNA that facilitated HCC metastasis by sponging miR-122-5p to regulate the expression of TGFBR2, SMAD2, and SMAD3. Further, TGF-β1 could enhance the expression of LINC01094, forming a positive feedback loop. TGF-β1-induced LINC01094 expression promotes HCC cell migration and invasion by targeting the miR-122-5p/TGFBR2-SMAD2-SMAD3 axis. LINC01094 may be a potential prognostic biomarker and therapeutic target for HCC metastasis.

Epigenetically associated IGF2BP3 upregulation promotes cell proliferation by regulating E2F1 expression in hepatocellular carcinoma

RNA-binding proteins (RBPs) are a class of proteins that primarily function by interacting with different types of RNAs and play a critical role in regulating the transcription and translation of cancer-related genes. However, their role in the progression of hepatocellular carcinoma (HCC) remains unclear. In this study, we analyzed RNA sequencing data and the corresponding clinical information of patients with HCC to screen for prognostic RBPs. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) was identified as an independent prognostic factor for liver cancer. It is upregulated in HCC and is associated with a poor prognosis. Elevated IGF2BP3 expression was validated via immunohistochemical analysis using a tissue microarray of patients with HCC. IGF2BP3 knockdown inhibited the proliferation of Hep3B and HepG2 cells, whereas IGF2BP3 overexpression promoted the expansion of HuH-7 and MHCC97H cells. Mechanistically, IGF2BP3 modulates cell proliferation by regulating E2F1 expression. DNA hypomethylation of the IGF2BP3 gene may increase the expression of IGF2BP3, thereby enhancing cell proliferation in HCC. Therefore, IGF2BP3 may act as a novel prognostic biomarker and a potential therapeutic target for HCC.