Primary Hepatocellular Carcinoma Cells Research Articles

Exposure of benzo[a]pyrene induces HCC exosome-circular RNA to activate lung fibroblasts and trigger organotropic metastasis.

Benzo[a]pyrene (B[a]P), a carcinogen pollutant produced by combustion processes, is present in the western diet with grilled meats. Chronic exposure of B[a]P in hepatocellular carcinoma (HCC) cells promotes metastasis rather than primary proliferation, implying an unknown mechanism of B[a]P-induced malignancy. Given that exosomes carry bioactive molecules to distant sites, we investigated whether and how exosomes mediate cancer-stroma communications for a toxicologically associated microenvironment. Exosomes were isolated from B[a]P stimulated BEL7404 HCC cells (7404-100Bap Exo) at an environmental relevant dose (100 nmol/L). Lung pre-education animal model was prepared via injection of exosomes and cytokines. The inflammatory genes of educated lungs were evaluated using quantitative reverse transcription PCR array. HCC LM3 cells transfected with firefly luciferase were next injected to monitor tumor burdens and organotropic metastasis. Profile of B[a]P-exposed exosomes were determined by ceRNA microarray. Interactions between circular RNA (circRNA) and microRNAs (miRNAs) were detected using RNA pull-down in target lung fibroblasts. Fluorescence in situ hybridization and RNA immunoprecipitation assay was used to evaluate the "on-off" interaction of circRNA-miRNA pairs. We further developed an adeno-associated virus inhalation model to examine mRNA expression specific in lung, thereby exploring the mRNA targets of B[a]P induced circRNA-miRNA cascade. Lung fibroblasts exert activation phenotypes, including focal adhesion and motility were altered by 7404-100Bap Exo. In the exosome-educated in vivo model, fibrosis factors and pro-inflammatory molecules of are up-regulated when injected with exosomes. Compared to non-exposed 7404 cells, circ_0011496 was up-regulated following B[a]P treatment and was mainly packaged into 7404-100Bap Exo. Exosomal circ_0011496 were delivered and competitively bound to miR-486-5p in recipient fibroblasts. The down-regulation of miR-486-5p converted fibroblast to cancer-associated fibroblast via regulating the downstream of Twinfilin-1 (TWF1) and matrix metalloproteinase-9 (MMP9) cascade. Additionally, increased TWF1, specifically in exosomal circ_0011496 educated lungs, could promote cancer-stroma crosstalk via activating vascular endothelial growth factor (VEGF). These modulated fibroblasts promoted endothelial cells angiogenesis and recruited primary HCC cells invasion, as a consequence of a pre-metastatic niche formation. We demonstrated that B[a]P-induced tumor exosomes can deliver circ_0011496 to activate miR-486-5p/TWF1/MMP9 cascade in the lung fibroblasts, generating a feedback loop that promoted HCC metastasis.

Transcriptomic and genomic characteristics of intrahepatic metastases of primary liver cancer

BackgroundPatients with primary multifocal hepatocellular carcinoma (HCC) have a poor prognosis and often experience a high rate of treatment failure. Multifocal HCC is mainly caused by intrahepatic metastasis (IM), and though portal vein tumor thrombosis (PVTT) is considered a hallmark of IM, the molecular mechanism by which primary HCC cells invade the portal veins remains unclear. Therefore, it is necessary to recognize the early signs of metastasis of HCC to arrange better treatment for patients.ResultsTo determine the differential molecular features between primary HCC with and without phenotype of metastasis, we used the CIBERSORTx software to deconvolute cell types from bulk RNA-Seq based on a single-cell transcriptomic dataset. According to the relative abundance of tumorigenic and metastatic hepatoma cells, VEGFA+ macrophages, effector memory T cells, and natural killer cells, HCC samples were divided into five groups: Pro-T, Mix, Pro-Meta, NKC, and MemT, and the transcriptomic and genomic features of the first three groups were analyzed. We found that the Pro-T group appeared to retain native hepatic metabolic activity, whereas the Pro-Meta group underwent dedifferentiation. Genes highly expressed in the group Pro-Meta often signify a worse outcome.ConclusionsThe HCC cohort can be well-typed and prognosis predicted according to tumor microenvironment components. Primary hepatocellular carcinoma may have obtained corresponding molecular features before metastasis occurred.

Abstract 5927: ACBP/DBI inhibition suppresses NASH-induced hepatocarcinogenesis

Abstract Background: Non-alcoholic fatty liver disease (NAFLD) or its severe form non-alcoholic steatohepatitis (NASH) is becoming an ever more prevalent risk factor of hepatocellular carcinoma (HCC). Currently, there are no approved drugs for the treatment of NASH. Extracellular ACBP/DBI (Acyl coenzyme A binding protein, also called diazepam-binding inhibitor) acts as an autophagy checkpoint and neutralization of ACBP/DBI prevents NASH. Therefore, we investigated the functional roles, mechanisms, and clinical relevance of ACBP/DBI in HCC. Methods: ACBP/DBI was blocked by four strategies: an inducible Acbp/Dbi knockout (Acbp-/-, control: Acbp+/+), a constitutive Gabrg2F77I mutation that prevents binding of ACBP/DBI to the receptor (control: WT), induction of ACBP/DBI-specific autoantibodies (induced by KLH-DBI, control: KLH), and monoclonal anti-DBI antibody (control: isotype). Three different types of HCC mouse models, including toxins/diet-based NASH-induced models (western diet plus CCl4), hydrodynamic transfection of oncogenes (Myc+Ctnnb1), and orthotopic transplantation (Hep55.1C) were used in combination with the aforementioned ACBP/DBI inhibition strategies to determine the association of ACBP/DBI and HCC. Results: ACBP/DBI inhibition attenuates tumorigenesis in mice with NASH, blunts oncogene induced HCC, and reduces the growth of orthotopically transplanted cancers. Mechanistically, ACBP/DBI inhibition retards cell cycle, suppresses cell proliferation, and favors ferroptosis, as determined by RNA-seq data and mass spectrometric metabolomics. CCK-8 proliferation, colony formation, and cell cycle assays performed on Huh-7, HepG2 and Hep55.1C cells demonstrated that ACBP/DBI knockdown reduced cell proliferation and induced G0/G1 arrest. IHC detection of Ki67 and PCNA in liver sections revealed antiproliferative effects of ACBP/DBI inhibition. qRT-PCR demonstrated the ACBP/DBI inhibition-induced upregulation of cell cycle inhibitor genes and downregulation of genes invoded in G0/G1 phase in liver tissues. qRT-PCR and immunoblot confirmed the upregulation of pro-ferroptotic and the downregulation of anti-ferroptotic genes and proteins in liver tissues upon ACBP/DBI inhibition. Primary HCC cell lines from Acbp+/+ and Acbp-/- mice further confirmed the pro-ferroptotic effects of ACBP/DBI inhibition obtained in liver tissues. Acbp-/- cell lines were more sensitivity to RSL3-induced ferroptosis. Conclusion: ACBP/DBI inhibition constitutes a potential strategy for treating NASH and NASH-induced HCC. Citation Format: Sijing Li, Omar Motiño, Flavia Lambertucci, Maria Chiara Maiuri, Isabelle Martins, Guido Kroemer. ACBP/DBI inhibition suppresses NASH-induced hepatocarcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5927.

Quercetin-induced degradation of RhoC suppresses hepatocellular carcinoma invasion and metastasis.

Tumor metastasis and recurrence are major causes of mortality in patients with hepatocellular carcinoma (HCC) that is still lack of effective therapeutic targets and drugs. Previous reports implied that ras homolog family member C (RhoC) plays a toxic role on metastasis and proliferation of cancer. In this research, the correlation between RhoC and metastasis ability was confirmed by invitro experiments and TCGA database. We explored whether quercetin could inhibit cell migration or invasion by transwell assay. Real-time PCR, overexpression and ubiquitination assay, etc. were applied in mechanism study. Primary HCC cells and animal models including patient-derived xenografts (PDXs) were employed to evaluate the anti-metastasis effects of quercetin. Clinical relevance and in vitro experiments further confirmed the level of RhoC was positively correlated with invasion and metastasis ability of HCC. Then we uncovered that quercetin could attenuate invasion and metastasis of HCC by downregulating RhoC's level in vitro, in vivo and PDXs. Furthermore, mechanistic investigations displayed quercetin hindered the E3 ligase expression of SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) leading to enhancement of RhoC's ubiquitination and proteasomal degradation. Our research has revealed the novel mechanisms quercetin regulates degradation of RhoC level by targeting SMURF2 and identified quercetin may be a potential compound for HCC therapy.

Efficacy of photodynamic therapy using hematoporphyrin derivative nanomedicine on hepatocellular carcinoma cells.

Objective: To demonstrate the efficacy of photodynamic therapy (PDT) using hematoporphyrin derivative (HPD) nanomedicine in combination with conventional chemotherapy based on gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) magnetic resonance imaging (MRI) for hepatocellular carcinoma (HCC) therapy. Methods: HPD nanomedicine was prepared, and the cytotoxicity of HPD nanomedicine at different concentrations on HCC cells and the half-maximal inhibitory concentration (IC50) were analyzed. Sixty HCC patients who visited our hospital from 2021 to 2023 were retrospectively analyzed. Patient data were analyzed, with 30 cases in control group (CG) receiving conventional chemotherapy for HCC, and 30 cases in observation group (OG) receiving conventional chemotherapy combined with HPD nanomedicine PDT. Gd-DTPA MRI was utilized to monitor the morphological and biological characteristics of the lesions in patients. After treatment completion, the long-term efficacy of patients and the levels of bcl-2 and bax proteins in primary HCC cells were evaluated. Results: The IC50 values of HPD on HepG2 cell proliferation and the cell inhibition rates gradually increased with increasing doses of HPD (50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM, 0.78 μM). HPD exhibited great anti-proliferative effects on HepG2 cells at relatively low concentrations. The differences in expression rates of bcl-2 protein and bax protein between groups were considerable (P<0.05). There were neglectable changes in AST and ALT levels between the two groups before treatment, but they were markedly reduced after treatment versus before treatment (P<0.05), with OG showing considerably lower levels than CG after treatment (P<0.05). Additionally, patients in OG exhibited better survival rates after the course of treatment versus those in CG. Conclusion: This study demonstrates that the combination of conventional chemotherapy based on Gd-DTPA MRI with HPD nanomedicine PDT greatly improves the efficacy of treatment for HCC patients. This combined treatment strategy not only enhances therapeutic outcomes but also alleviates adverse reactions associated with conventional treatment, providing a novel approach for future research in the treatment of HCC.

Artificial Intelligence-assisted Raman Spectroscopy for Liver cancer diagnosis

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, represents a global health challenge due to its complexity and the limitations of current diagnostic techniques. By combining Raman spectroscopy and Artificial Intelligence (AI), we have succeeded in classifying tumor cells. In fact, we have performed a first Raman spectral analysis based on the characterization and differentiation between uncultured primary human liver cells derived from resected HCC tumor tissue and the adjacent non-tumor counterpart. Biochemical analysis of the collected Raman spectra revealed that there is more DNA in the nuclei of the tumor cells than in non-tumor cells. We then develop three machine learning approaches, including multivariate models and neural networks, to rapidly automate the recognition and classification of the Raman spectra of both cells. To evaluate the performance of the developed AI models, we prepared and analyzed two additional cell samples with a ratio of 4:1 and 3:1 between tumor and non-tumor cells and compared the obtained results with the nominal percentages (accuracy of 80 and 60%, respectively). These results confirm that the models are able to make classifications at the level of a single spectrum, indicating the possibility of rapidly analysing and classifying a primary HCC cell.

Generating Patient-Derived HCC Cell Lines Suitable for Predictive In Vitro and In Vivo Drug Screening by Orthotopic Transplantation.

Hepatocellular carcinoma (HCC) results in high mortality due to ineffective systemic therapy. Human immortalized cell lines are commonly used to study anti-tumor effects in the context of new anti-tumor therapies and tumor biology. As immortalized cell lines have limited biological relevance and heterogeneity compared to primary cells, patient-derived tumor tissues, and corresponding immune cells are the gold standards for studying the complexity of individual tumor entities. However, culturing primary HCC cells has a low success rate. Here, we aimed to establish a reproducible approach to preserve the patient-derived liver cancer cells for in vitro and in vivo studies. The underlying study aimed to establish an in vitro pre-screening platform to test treatment options' effectivity and dosage, e.g., for new substances, autologous modified immune cells, or combined therapies in HCC. We initially employed 15 surgical resection specimens from patients with different HCC entities for isolation and preservation. The isolated liver cancer cells from four HCC-diagnosed patients were used for orthotopic transplantation into the healthy liver of immunodeficient mice, allowing them to grow for six months before human liver cancer cells were isolated and cultured. As a result, we generated and characterized four new primary-like liver cancer cell lines. Compared to immortalized HCC cell lines, freshly generated liver cancer cells displayed individual morphologies and heterogeneous protein-level characteristics. We assessed their ability to proliferate, migrate, form spheroids, and react to common medications compared to immortalized HCC cell lines. All four liver cancer cell lines exhibit strong migration and colony-forming characteristics in vitro, comparable to extensively investigated immortalized HCC cell lines. Moreover, the four etiological different liver cancer cell lines displayed differences in the response to 5-FU, Sorafenib, Axitinib, and interferon-alpha treatment, ranking from non-responders to responders depending on the applicated medication. In sum, we generated individual patient-derived liver cancer cell lines suitable for predictive in vitro drug screenings and for xenograft transplantations to realize the in vivo investigation of drug candidates. We overcame the low cultivation success rate of liver cancer cells derived from patients and analyzed their potential to serve a pre-clinical model.

LncRNA SNHG9 enhances liver cancer stem cell self-renewal and tumorigenicity by negatively regulating PTEN expression via recruiting EZH2.

Liver cancer stem cell (CSC) self-renewal and tumorigenesis are important causes of hepatocellular carcinoma (HCC) recurrence. We purposed to investigate the function of long noncoding RNA small nucleolar RNA host gene 9 (SNHG9) in liver CSC self-renewal and tumorigenesis in this study. Flow cytometry was carried out to separate CD133+ Populations and CD133- Populations from HCC cell lines. A combination of CD133+ cells and Matrigel matrix was subcutaneously injected to create the NOD-SCID mouse xenograft tumor model. Colony formation test and spheroids formation assay were carried out to clarify the impact of SNHG9 on the self-renewal of liver CSCs. RNA immunoprecipitation, RNA-pull down, and chromatin immunoprecipitation were performed on CD133+ cells to elucidate the mechanism of SNHG9 regulating PTEN expression. We found that SNHG9 was highly expressed in HCC clinical samples, HCC cells, and CD133+ cells. In vitro, interference with SNHG9 prevented the formation of colonies and spheroids in liver CSC cells and primary HCC cells. In vivo, interference with SNHG9 reduced the tumor volume and weight. SNHG9 could bind to EZH2, and SNHG9 interference suppressed EZH2 recruitment and H3K27me3 levels in the PTEN promoter region. In addition, SNHG9 inhibition promoted PTEN expression while having little impact on EZH2 levels. Interference with SNHG9 inhibited liver CSC self-renewal and tumorigenesis by up-regulating PTEN levels. In conclusion, by binding to EZH2, SNHG9 down-regulated PTEN levels, promoting liver CSC self-renewal and tumor formation, and exacerbating HCC progression.

Long Non-coding RNA LINC00342 Promotes the Proliferation, Invasion, and Migration of Primary Hepatocellular Carcinoma Cells by Regulating the Expression of miRNA-19a-3p, miRNA-545-5p, and miRNA-203a-3p.

This study aimed to investigate the role of the long non-coding RNA (lncRNA) LINC00342-207 (LINC00342) in the development and progression of primary hepatocellular carcinoma (HCC). Forty-two surgically resected HCC tissues and corresponding paracancerous tissues were collected from October 2019 to December 2020 and examined for lncRNA LINC00342, microRNA (miR)-19a-3p, miR-545-5p, miR-203a-3p, cell cycle protein D1 (CyclinD1/CCND1), murine double minute 2 (MDM2), and fibroblast growth factor 2 (FGF2) expression. The disease-free survival and overall survival of patients with HCC were followed up. HCC cell lines and the normal hepatocyte cell line HL-7702 were cultured and the expression level of LINC00342 was measured. HepG2 cells were transfected with LINC00342 siRNA, LINC00342 overexpression plasmid, miR-19a-3p mimics and their corresponding suppressors, miR-545-5p mimics and their corresponding suppressors, and miR-203a-3p mimics and their corresponding suppressors. The proliferation, apoptosis, migration, and invasion of HepG2 cells were detected. Stably transfected HepG2 cells were inoculated into the left axilla of male BALB/c nude mice, and the volume and quality of transplanted tumors as well as the expression levels of LINC00342, miR-19a-3p, miR-545-5p, miR-203a-3p, CCND1, MDM2, and FGF2 were examined. LINC00342 played an oncogenic role in HCC and exhibited inhibitory effects on proliferation, migration, and invasion, and promoted the apoptosis of HepG2 cells. Moreover, it inhibited the growth of transplanted tumors in vivo in mice. Mechanistically, the oncogenic effect of LINC00342 was associated with the targeted regulation of the miR-19a-3p/CCND1, miR-545-5p/MDM2, and miR-203a-3p/FGF2 axes.

Inhibitory effects of diarsenic trioxide (As2O3) on hepatocellular carcinoma cells exerted by regulation of promyelocytic leukemia protein levels

Previous Chinese research revealed that diarsenic trioxide (As2O3) inhibits acute promyelocytic leukemia (PML) cell proliferation and initiates apoptosis through degradation of the PML-retinoic acid receptor protein. This study was to analyse whether As2O3 also had an effect on hepatocellular carcinoma (HCC) cells. As2O3 effects on various HCC cell lines and primary HCC cells were investigated in time and dose series, including measurements of cell growth, PML mRNA and protein expression, xenografted tumor formation, and the self-renewal Oct4 and hepatocyte marker expressions in mouse model xenografts or cells treated with PML siRNA. The results were analyzed by immunocytochemistry, quantitative reverse transcription PCR and western blotting as well as indocyanine green and Periodic Acid Schiff staining. As2O3 inhibited HCC cell and HCC cell-derived xenograft tumor formation in a time-dependent manner and reduced PML protein expression in HCC cells, but had limited effects on PML mRNA levels in cell nuclei. The HCC cell line HuH7 treated with As2O3 showed a decreased expression of alpha-fetoprotein and increased expression and transcription of mature hepatocyte markers, indicating differentiation of HCC cells into hepatocytes. Cytokeratin 18 protein and mRNA levels as well as tyrosine aminotransferase and apolipoprotein B mRNA transcriptions were enhanced by As2O3 as were the numbers of indocyanine green and Periodic Acid Schiff stained cells. In addition, As2O3 downregulated the expression of Oct4. In conclusion, since As2O3 inhibited HCC cell proliferation and HCC cell-derived xenograft tumor formation it is suggested that an appropriate concentration of As2O3 might be a promising therapy to treat HCC.

Dehydroepiandrosterone protects against oleic acid-triggered mitochondrial dysfunction to relieve oxidative stress and inflammation via activation of the AMPK-Nrf2 axis by targeting GPR30 in hepatocytes.

Fatty liver hemorrhage syndrome (FLHS) seriously threatens the health and performance of laying hens, and the occurrence and development of FLHS are closely related to oxidative damage and inflammation; thus, diets supplemental with activated substances to relive the oxidative stress and inflammation maybe effectively control the occurrences of FLHS. Dehydroepiandrosterone (DHEA) has beneficial effects in fat-reduction, anti-oxidation and anti-inflammation, and it was widely applied to alleviate multiple metabolic-related diseases; however, there are few reports on whether DHEA can prevent against metabolic-related diseases by modulating oxidative stress and inflammation, especially FLHS in laying hens. Herein, present study aimed to investigate the regulatory actions and potential molecular mechanism of DHEA on inflammation and oxidative stress triggered by oleic acid (OA)-stimulation in primary chicken hepatocytes and chicken hepatocellular carcinoma cell line (LMH). The results showed that DHEA significantly alleviated oxidative stress challenged by OA-stimulation via activation of AMP-activated protein kinase (AMPK)-nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling pathway in hepatocytes, which led to relieving effect of DHEA on inflammatory by inhibiting mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) signaling pathways. Mechanistically, we found that the activation of AMPK-Nrf2 signaling pathway by DHEA treatment was mediated by G-protein coupled estrogen receptor (GPR30/GPER) in OA-stimulated hepatocytes. Further investigation found that DHEA activated the GPR30-mediated AMPK-Nrf2 signaling pathways to increase antioxidant capacity and inhibit mitochondrial reactive oxygen species (ROS) overproduction, which thereby inhibiting the activation of ROS-induced MAPK and NF-κB signaling pathways in OA-stimulated hepatocytes. Overall, these data demonstrated that DHEA attenuates the oxidative stress and inflammation triggered by OA-stimulation, and these beneficial effects of DHEA are achieved by activating the GPR30-mediated AMPK-Nrf2 signaling to prevent the impairment of mitochondrial function, and thereby inhibiting the activation of ROS-induced MAPK and NF-κB signaling pathways in hepatocytes. These results revealed the effects and mechanisms of DHEA on oxidative stress and inflammation, and also provide substantial information to support it as a potential nutritional supplement in preventing the occurrences of FLHS in laying hens and other metabolic-related diseases in animals and humans.

Identification of G protein subunit alpha i2 as a promising therapeutic target of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a global health problem. Its incidence and mortality are increasing. Exploring novel therapeutic targets against HCC is important and urgent. We here explored the expression and potential function of Gαi2 (G protein subunit alpha i2) in HCC. The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) database shows that the number of Gαi2 transcripts in HCC tissues is significantly higher than that in the normal liver tissues. Moreover, Gαi2 overexpression in HCC correlates with poor prognosis of the patients. Gαi2 mRNA and protein expression are also elevated in local HCC tissues and different human HCC cells. In patient-derived primary HCC cells and immortalized HepG2 cells, Gαi2 silencing (by targeted shRNA) or knockout (KO, by the dCas9-sgRNA method) largely suppressed cell proliferation and motility, while inducing cell cycle arrest and caspase-apoptosis activation. Moreover, Gαi2 silencing or KO-induced reactive oxygen species (ROS) production and oxidative injury in primary and HepG2 HCC cells. Whereas different antioxidants ameliorated Gαi2-shRNA-induced anti-HCC cell activity. Using a lentiviral construct, Gαi2 overexpression further augmented proliferation and motility of primary and immortalized HCC cells. Further studies revealed that the binding between the transcription factor early growth response zinc finger transcription factor 1 (EGR1) and Gαi2 DNA promoter was significantly increased in HCC tissues and cells. In vivo, intratumoral injection of Gαi2 shRNA adeno-associated virus significantly hindered HCC xenograft growth in nude mice. Moreover, the growth of Gαi2-KO HCC xenografts in the nude mice was remarkably slow. Gαi2 depletion, oxidative injury, and apoptosis induction were detected in Gαi2-silenced or Gαi2-KO HCC xenografts. Together, overexpressed Gαi2 is required for HCC cell growth in vitro and in vivo, representing as a novel and promising diagnosis marker and therapeutic target of HCC.

LncRNA SLC1A5-AS/MZF1/ASCT2 Axis Contributes to Malignant Progression of Hepatocellular Carcinoma.

Hypoxia is a pivotal factor influencing cellular gene expression and contributing to the malignant progression of tumors. Metabolic anomalies under hypoxic conditions are predominantly mediated by mitochondria. Nonetheless, the exploration of hypoxia-induced long noncoding RNAs (lncRNAs) associated with mitochondria remains largely uncharted. We established hypoxia cell models using primary human hepatocytes (PHH) and hepatocellular carcinoma (HCC) cell lines. We isolated mitochondria for high-throughput sequencing to investigate the roles of candidate lncRNAs in HCC progression. We employed in vitro and in vivo assays to evaluate the functions of solute carrier family 1 member 5 antisense lncRNA (SLC1A5-AS). RNA-seq was utilized to scrutinize the comprehensive genome profile regulated by SLC1A5-AS in HCC. Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis were utilized to validate the expression of alanine-serine-cysteine transporter 2 (ASCT2, encoded by the SLC1A5 gene), and a glutamine uptake assay was employed to estimate the glutamine uptake capacity of Huh-7 cells after SLC1A5-AS overexpression. To delve into the mechanisms governing the regulation of SLC1A5 expression by SLC1A5-AS, we employed a biotin-labeled SLC1A5-AS probe in conjunction with a western blot assay to confirm the interactions between SLC1A5-AS and candidate transcription factors. Luciferase reporter assays and chromatin immunoprecipitation (ChIP) were utilized to authenticate the effects of the predicted transcription factors on SLC1A5 promoter activity. Following the screening, we identified CTB-147N14.6, derived from the antisense strand of the SLC1A5 gene, which we have named SLC1A5-AS. SLC1A5-AS exhibited significantly elevated expression levels in HCC tissue and was associated with poor prognosis in HCC patients. In vitro and in vivo assays revealed that the overexpression of SLC1A5-AS significantly heightened cell invasion and metastasis. RNA-seq data unveiled SLC1A5-AS involvement in glutamine metabolism, left-handed amino (L-amino) acid transmembrane transporter activity, and the nuclear factor kappa-B (NF-κB) signaling pathway. Overexpression of SLC1A5-AS markedly increased ASCT2 mRNA/protein levels, thereby enhancing glutamine uptake and promoting the growth and metastasis of HCC cells. Mechanistically, higher RNA levels of SLC1A5-AS directly bound with myeloid zinc finger 1 (MZF1), acting as a transcriptional repressor, thus diminishing its binding to the SLC1A5 promoter region. Our findings unveil a novel role for the lncRNA SLC1A5-AS in glutamine metabolism, suggesting that targeting SLC1A5-AS/MZF1, in conjunction with ASCT2 inhibitor treatment, could be a potential therapeutic strategy for this disease.

Anti-Cancer Effects of YAP Inhibitor (CA3) in Combination with Sorafenib against Hepatocellular Carcinoma (HCC) in Patient-Derived Multicellular Tumor Spheroid Models (MCTS).

Simple SummaryActivation of YAP/TAZ (mediators of Hippo signaling) is associated with the development of liver cancer. The expression level of YAP is known to relate to chemoresistance. However, the therapeutic effect of YAP/TAZ inhibition when combined with sorafenib and conventional chemotherapy in HCC is not known. Recent studies have highlighted the importance of the tumor microenvironment (TME) in chemoresistance. The multicellular tumor spheroid (MCTS) model has emerged as a promising tool for studying cancer drugs as it mimics actual TME. Here, we aimed at assessing the YAP/TAZ expression level of HCCs and identifying the therapeutic effects of CA3 (novel YAP inhibitor) when combined with sorafenib using a patient-derived MCTS model. We established patient-derived MCTS and confirmed that patient-derived MCTS with high YAP/TAZ expression responded more sensitively to the combination therapy (sorafenib with CA3) than MCTS with low or medium YAP/TAZ expression. These findings suggest that the YAP/TAZ inhibitor may serve as a potential therapeutic strategy to enhance sensitivity to sorafenib in HCCs with high YAP/TAZ expression.Purpose: To assess the expression levels of YAP and TAZ in patient-derived HCC tissue and identify the effects of YAP/TAZ inhibition depending on the baseline YAP/TAZ expression when combined with sorafenib using a patient-derived multicellular tumor spheroid (MCTS) model. Methods: Primary HCC cell lines were established from patient-derived tissue. Six patient-derived HCC cell lines were selected according to YAP/TAZ expression on Western blot: high, medium, low. Then, MCTS was generated by mixing patient-derived HCC cells and stroma cells (LX2, WI38, and HUVECs) and YAP/TAZ expression was assessed using Western blot. Cell viability of MCTS upon 48 h of drug treatment (sorafenib, sorafenib with CA3 0.1 µM, and CA3 (novel YAP1 inhibitor)) was analyzed. Results: Out of six patient-derived HCC cell lines, cell lines with high YAP/TAZ expression at the MCTS level responded more sensitively to the combination therapy (Sorafenib + CA3 0.1 μM) despite the potent cytotoxic effect of CA3 exhibited in all of the patient-derived HCCs. Conclusion: Targeting YAP/TAZ inhibition using the novel YAP1 inhibitor CA3 could be a promising therapeutic strategy to enhance sensitivity to sorafenib especially in HCCs with high YAP/TAZ expression in MCTS.

In Vivo Screen Identifies Liver X Receptor Alpha Agonism Potentiates Sorafenib Killing of Hepatocellular Carcinoma.

In Vivo Screen Identifies Liver X Receptor Alpha Agonism Potentiates Sorafenib Killing of Hepatocellular Carcinoma.

ITGB1 Drives Hepatocellular Carcinoma Progression by Modulating Cell Cycle Process Through PXN/YWHAZ/AKT Pathways.

Integrin β1 (ITGB1), which acts as an extracellular matrix (ECM) receptor, has gained increasing attention as a therapeutic target for the treatment of hepatocellular carcinoma (HCC). However, the underpinning mechanism of how ITGB1 drives HCC progression remains elusive. In this study, we first found that ITGB1 expression was significantly higher in HCC tissues than in normal controls by bioinformatics analysis. Furthermore, bioinformatics analysis revealed that paxillin (PXN) and 14-3-3 protein zeta (YWHAZ) are the molecules participating in ITGB1-regulated HCC tumor cell cycle progression. Indeed, immunohistochemistry (IHC) revealed that ITGB1, paxillin, and YWHAZ were strongly upregulated in paired HCC tissue compared with adjacent normal tissues. Notably, the inhibition of ITGB1 expression by small interfering RNA (siRNA) resulted in the downregulated expression of PXN and YWHAZ in primary HCC cells, as assessed by western blot and immunostaining. In addition, ITGB1 knockdown markedly impaired the aggressive behavior of HCC tumor cells and delayed cell cycle progression as determined by cell migration assay, drug-resistance analysis, colony formation assay, quantitative real-time polymerase chain reaction (qRT-PCR), and cell cycle analysis as well as cell viability measurements. More importantly, we proved that xenograft ITGB1high tumors grew more rapidly than ITGB1low tumors. Altogether, our study showed that the ITGB1/PXN/YWHAZ/protein kinase B (AKT) axis enhances HCC progression by accelerating the cell cycle process, which offers a promising approach to halt HCC tumor growth.

Antitumor Effects of Scorpion Peptide Smp43 through Mitochondrial Dysfunction and Membrane Disruption on Hepatocellular Carcinoma.

Smp43, a cationic antimicrobial peptide identified from the venom gland of the Egyptian scorpion Scorpio maurus palmatus, shows cytotoxicity toward hepatoma cell line HepG2 by membrane disruption. However, its underlying detailed mechanisms still remain to be further clarified. In the present study, we evaluated the cellular internalization of Smp43 and explored its effects on cell viability, cell cycle, apoptosis, autophagy, necrosis, and factor expression related to these cellular processes in human HepG2. Smp43 was found to suppress the growth of HepG2, Huh7, and human primary hepatocellular carcinoma cells while showing low toxicity to normal LO2 cells. Furthermore, Smp43 could interact with the cell membrane and be internalized into HepG2 cells via endocytosis and pore formation, which caused a ROS production increase, mitochondrial membrane potential decline, cytoskeleton disorganization, dysregulation of cyclin expression, mitochondrial apoptotic pathway activation, and alteration of MAPK as well as PI3K/Akt/mTOR signaling pathways. Finally, Smp43 showed effective antitumor protection in the HepG2 xenograft mice model. Overall, these findings indicate that Smp43 significantly exerts antitumor effects via induction of apoptosis, autophagy, necrosis, and cell cycle arrest due to its induction of mitochondrial dysfunction and membrane disruption. This discovery will extend the antitumor mechanisms of antimicrobial peptides and contribute to the development of antitumor agents against hepatocellular carcinoma.

Complex Tumor Spheroid Formation and One-Step Cancer-Associated Fibroblasts Purification from Hepatocellular Carcinoma Tissue Promoted by Inorganic Surface Topography.

In vitro cell models play important roles as testbeds for toxicity studies, drug development, or as replacements in animal experiments. In particular, complex tumor models such as hepatocellular carcinoma (HCC) are needed to predict drug efficacy and facilitate translation into clinical practice. In this work, topographical features of amorphous silicon dioxide (SiO2) are fabricated and tested for cell culture of primary HCC cells and cell lines. The topographies vary from pyramids to octahedrons to structures named fractals, with increased hierarchy and organized in periodic arrays (square or Hexagonal). The pyramids were found to promote complex 2D/3D tissue formation from primary HCC cells. It was found that the 2D layer was mainly composed of cancer-associated fibroblasts (CAFs), while the 3D spheroids were composed of tumor cells enwrapped by a CAF layer. Compared with conventional protocols for 3D cultures, this novel approach mimics the 2D/3D complexity of the original tumor by invading CAFs and a microtumor. Topographies such as octahedrons and fractals exclude tumor cells and allow one-step isolation of CAFs even directly from tumor tissue of patients as the CAFs migrate into the structured substrate. Cell lines form spheroids within a short time. The presented inorganic topographical surfaces stimulate complex spheroid formation while avoiding additional biological scaffolds and allowing direct visualization on the substrate.

Hepatotoxicity of Pyrrolizidine Alkaloid Compound Intermedine: Comparison with Other Pyrrolizidine Alkaloids and Its Toxicological Mechanism.

Pyrrolizidine alkaloids (PAs) are common secondary plant compounds with hepatotoxicity. The consumption of herbal medicines and herbal teas containing PAs is one of the main causes of hepatic sinusoidal obstruction syndrome (HSOS), a potentially life-threatening condition. The present study aimed to reveal the mechanism underlying the cytotoxicity of intermedine (Im), the main PA in Comfrey. We evaluated the toxicity of the retronecine-type PAs with different structures to cell lines derived from mammalian tissues, including primary mouse hepatocytes, human hepatocytes (HepD), mouse hepatoma-22 (H22) and human hepatocellular carcinoma (HepG2) cells. The cytotoxicity of Im to hepatocyte was evaluated by using cell counting kit-8 assay, colony formation experiment, wound healing assay and dead/live fluorescence imaging. In vitro characterization showed that these PAs were cytotoxic and induced cell apoptosis in a dose-dependent manner. We also demonstrated that Im induced cell apoptosis by generating excessive reactive oxygen species (ROS), changing the mitochondrial membrane potential and releasing cytochrome c (Cyt c) before activating the caspase-3 pathway. Importantly, we directly observed the destruction of the cell mitochondrial structure after Im treatment through transmission electron microscopy (TEM). This study provided the first direct evidence of Im inducing hepatotoxicity through mitochondria-mediated apoptosis. These results supplemented the basic toxicity data of PAs and facilitated the comprehensive and systematic evaluation of the toxicity caused by PA compounds.

Combined inhibition of AURKA and HSF1 suppresses proliferation and promotes apoptosis in hepatocellular carcinoma by activating endoplasmic reticulum stress.

In this study we aimed to assess the anti-tumor effect of co-inhibition of Aurora kinase A (AURKA) and heat shock transcription factor 1 (HSF1) on hepatocellular carcinoma (HCC), as well as to explore the mechanism involved. Expression of AURKA and HSF1 in primary HCC tissues and cell lines was detected by immunohistochemistry (IHC), qRT-PCR and Western blotting. AURKA was knocked down in HepG2 and BEL-7402 HCC cells using lentivirus-mediated RNA interference. Next, CCK-8, clone formation, transwell and flow cytometry assays were used to assess their viability, migration, invasion and apoptosis, respectively. The expression of proteins related to cell cycle progression, apoptosis and endoplasmic reticulum stress (ERS) was analyzed using Western blotting. In addition, in vivo tumor growth of HCC cells was assessed using a nude mouse xenograft model, and the resulting tumors were evaluated using HE staining and IHC. Both AURKA and HSF1 were highly expressed in HCC tissues and cells, while being negatively related to HCC prognosis. Knockdown of AURKA significantly inhibited the colony forming and migrating capacities of HCC cells. In addition, we found that treatment with an AURKA inhibitor (Danusertib) led to marked reductions in the proliferation and migration capacities of the HCC cells, and promoted their apoptosis. Notably, combined inhibition of AURKA and HSF1 induced HCC cell apoptosis, while increasing the expression of ERS-associated proteins, including p-eIF2α, ATF4 and CHOP. Finally, we found that co-inhibition of AURKA and HSF1 elicited an excellent in vivo antitumor effect in a HCC mouse model with a relatively low cytotoxicity. Combined inhibition of AURKA and HSF1 shows an excellent anti-tumor effect on HCC cells in vitro and in vivo, which may be mediated by ERS. These findings suggest that both AURKA and HSF1 may serve as targets for HCC treatment.