Cell Lines Hep3B Research Articles

The association between several autophagy-related genes and their prognostic values in hepatocellular carcinoma: a study on the foundation of TCGA, GEPIA and HPA databases.

The purpose of this study was to investigate the relationship between the expression of autophagy-related genes and prognosis in hepatocellular carcinoma (HCC). We selected three autophagy-related genes (ATG3, ATG7, and ATG9A) from gene expression data of liver cancer patients in The Cancer Genome Atlas (TCGA) database by Kaplan-Meier survival analysis, univariate and multivariate Cox regression analysis, and Gene Set Enrichment Analysis (GSEA). Human Protein Atlas (HPA) and Gene Expression Profiling Interactive Analysis (GEPIA) databases were applied to testify the credibility of our results. The expression levels of ATG3, ATG7, and ATG9A were verified by real-time quantitative PCR (RT-qPCR) in normal liver cells (L02) and three HCC cell lines (HepG2, Hep3b, and Li-7). Data analysis results from TCGA showed high ATG3, ATG7, ATG9A expression in HCC tumor tissues. Kaplan-Meier survival analysis showed that the survival rate of the high expression group of ATG3, ATG7, and ATG9A was all significantly lower than the low expression group. GSEA analysis showed that many signaling pathways (such as the regulation of autophagy, glycine serine and threonine metabolism, pathways in cancer, mitogen-activated protein kinase (MAPK) signaling pathway, mammalian target of rapamycin (mTOR) signaling pathway, as well as P53 signaling pathway) were differentially enriched in HCCs with ATG3, ATG7, and ATG9A expression. GEPIA and RT-qPCR also identified that the mRNA expression level of ATG3, ATG7, and ATG9A in normal liver cells were significantly lower than in HCC cells. High protein expression of ATG3, ATG7, and ATG9A was displayed in HCCs from the HPA database. The ATG3, ATG7, ATG9A might be utilized as prognostic biomarkers for liver cancer.

COMMD3 Expression Affects Angiogenesis through the HIF1α/VEGF/NF-κB Signaling Pathway in Hepatocellular Carcinoma In Vitro and In Vivo

Background High expression of copper metabolizing MURR1 domain (COMMD3) is significantly correlated with poor prognosis in hepatocellular carcinoma (HCC) patients. Here, we explored the mechanism by which COMMD3 affects HCC angiogenesis through the HIF1α/VEGF/NF-κB signaling pathway. Methods SK-Hep1 and Hep-3B cell lines were transfected by COMMD3 overexpression and RNA interference lentivirus and verified using RT-qPCR and western blotting techniques. Using RNA sequencing, we analyzed differentially expressed genes in COMMD3-overexpressed and COMMD3-knockdown HCC cells. Altogether, colony formation assay, wound healing assay, transwell cell invasion assay, flow cytometry apoptosis experiments, HUVEC tube formation detection, phalloidin staining assay, western blotting, immunohistochemical staining, and a nude mouse xenograft model were used for experimental verification. Results Lentivirus COMMD3 overexpression and knockdown were successfully established in HCC cells. COMMD3 overexpression significantly promoted the proliferation, angiogenesis, migration, and invasion capacities of HCC cells with no obvious effect on apoptosis versus controls while COMMD3 knockdown showed the opposite trend. The expression and protein levels of COMMD3 as well as HIF1α, VEGF, and NF-κB were increased in COMMD3-overexpressing HCC cells versus control cells, while they were reduced after COMMD3 knockdown. In addition, RNA-seq indicated that COMMD3 is an indispensable gene for HCC angiogenesis through HIF1α and NF-κB signaling pathways. Conclusion This study showed that low expression of COMMD3 can inhibit HCC angiogenesis by suppressing the HIF1α/VEGF/NF-κB pathway. This implicates COMMD3 as a potential biomarker for improving the therapeutic outcome of HCC.

New Diterpenes with Potential Antitumoral Activity Isolated from Plants in the Years 2017-2022.

Diterpenes represent a wider class of isoprenoids, with more than 18,000 isolated compounds, and are present in plants, fungi, bacteria, and animals in both terrestrial and marine environments. Here, we report on the fully characterised structures of 251 new diterpenes, isolated from higher plants and published from 2017, which are shown to have antitumoral activity. An overview on the most active compounds, showing IC50 < 20 μM, is provided for diterpenes of different classes. The most active compounds were extracted from 29 different plant families; particularly, Euphorbiaceae (69 compounds) and Lamiaceae (54 compounds) were the richest sources of active compounds. A better activity than the positive control was obtained with 33 compounds against the A549 cell line, 28 compounds against the MCF-7 cell line, 9 compounds against the HepG2 cell line, 8 compounds against the Hep3B cell line, 19 compounds against the SMMC-7721 cell line, 9 compounds against the HL-60 cell line, 24 compounds against the SW480 cell line, and 19 compounds against HeLa.

Diaporine Potentiates the Anticancer Effects of Oxaliplatin and Doxorubicin on Liver Cancer Cells.

Recent studies have shown that diaporine, a novel fungal metabolic product, has a strong in vitro and in vivo anticancer effect on human non-small-cell lung and breast cancers. In this study, three human hepatocarcinoma cell lines (HepG2, Hep3B, and Huh7) were used to evaluate the efficacy of diaporine alone and in combination with the standard cytotoxic drugs oxaliplatin and doxorubicin for the treatment of liver cancer. We demonstrated that diaporine, oxaliplatin, and doxorubicin triggered a concentration- and time-dependent decrease in the number of HepG2 cells. Diaporine at a concentration of 2.5 μM showed almost 100% inhibition of cell counts at 72 h. Similar effects were observed only with much higher concentrations (100 μM) of oxaliplatin or doxorubicin. Decreases in cell numbers after 48 h treatment with diaporine, oxaliplatin, and doxorubicin were also demonstrated in two additional hepatoma cell lines, Hep3B and Huh7. The combination of these drugs at low concentration for 48 h in vitro noticeably showed that diaporine improved the inhibitory effect on the number of cancer cells induced by oxaliplatin or doxorubicin. Additionally, this combination effectively inhibited colony growth in vitro. We found that inhibition of phosphorylation of ERK1/2 significantly increased when diaporine was used in combination with other agents. In addition, we also found that when diaporine was used in combination with doxorubicin or oxaliplatin, their proapoptotic effect greatly increased. We further revealed that the induction of apoptosis in hepatoma cells after treatment is due, at least in part, to the inhibition of phosphorylation of AKT, leading to the activation of caspase-3, inactivation of poly (ADP-ribose) polymerase (PARP), and subsequently to DNA damage, as indicated by the increased level of H2AX. Based on these findings, we suggest that diaporine in combination with the standard cytotoxic drugs oxaliplatin and doxorubicin may play a role in the treatment of liver cancer.

Preclinical Characterization of Vadadustat (AKB-6548), an Oral Small Molecule Hypoxia-Inducible Factor Prolyl-4-Hydroxylase Inhibitor, for the Potential Treatment of Renal Anemia.

Pharmacological inhibition of prolyl-4-hydroxylase domain (PHD) enzymes stabilizes hypoxia-inducible factors (HIFs), transcription factors that activate target genes that, among others, increase erythropoietin (EPO) synthesis, resulting in the production of new red blood cells (RBCs). Herein, we summarize the preclinical characteristics of the small molecule HIF prolyl-4-hydroxylase inhibitor vadadustat (AKB-6548), which is in development for the treatment of anemia in patients with chronic kidney disease (CKD). Vadadustat inhibits the enzyme activity of all three human PHD isozymes, PHD1, PHD2, and PHD3, with similar low nanomolar inhibitory constant values. PHD enzyme inhibition by vadadustat is competitive with endogenous cofactor 2-oxoglutarate and is insensitive to free iron concentration. In the human hepatocellular carcinoma cell line (Hep 3B) and human umbilical vein endothelial cells, PHD inhibition by vadadustat leads to the time- and concentration-dependent stabilization of HIF-1α and HIF-2α In Hep 3B cells, this in turn results in the synthesis and secretion of EPO; vascular endothelial growth factor is not measured at detectable levels. A single oral dose of vadadustat in rats potently increases circulating levels of EPO, and daily oral dosing for 14 days increases RBC indices in healthy rats and in the 5/6 nephrectomy model of CKD. In mice and dogs, once-daily repeat oral dosing increases hemoglobin and hematocrit. Vadadustat has a relatively short half-life in all nonclinical species evaluated and does not accumulate when administered as a single bolus dose (oral or intravenous) or upon repeat oral dosing. The pharmacological profile of vadadustat supports continued development for treatment of renal anemia. SIGNIFICANCE STATEMENT: Vadadustat (AKB-6548) is an orally bioavailable small molecule prolyl-4-hydroxylase inhibitor in development for anemia of chronic kidney disease. It is an equipotent inhibitor of the three human prolyl-4-hydroxylase domain isoforms, which activates erythropoiesis through stabilization of hypoxia-inducible factor (HIF)-1α and HIF-2α, increasing production of erythropoietin, without detectable stimulation of vascular endothelial growth factor.

Leucine zipper downregulated in cancer 1 may serve as a favorable prognostic biomarker by influencing proliferation, colony formation, cell cycle, apoptosis, and migration ability in hepatocellular carcinoma.

Aims: Leucine zipper downregulated in cancer 1 (LDOC1) inhibits tumor growth in several cancers. However, the expression and function of LDOC1 in hepatocellular carcinoma (HCC) remain unknown. In this study, we aimed to investigate how LDOC1 influenced tumor progression and the biological functions of HCC. Methods: The transcription levels of LDOC1 were determined using the GEPIA and UALCAN online databases and a real-time polymerase chain reaction. Western blot and immunohistochemistry were used to validate the protein levels of LDOC1. The online Kaplan-Meier Plotter was applied for survival analysis. Then lentivirus transfection was used to construct LDOC1 exogenous overexpression cell lines. Proliferation, clone formation, cell cycle, apoptosis, and migration assays were performed with the LDOC1-upregulated Huh7 and Hep3B cell lines. The phosphorylated and total levels of AKT and mTOR were determined using a Western blot to explore the potential molecular mechanism of LDOC1. Results: In the GEPIA and UALCAN analyses, LDOC1 was lowly expressed in tumors, had high expression in normal tissue samples (p < 0.05), and negatively correlated with tumor grade progression. The down-regulation of LDOC1 in HCC was validated with real-time polymerase chain reaction, Western blot, and immunohistochemistry (all p < 0.05). LDOC1 transcription levels were negatively associated with overall, progression-free, recurrence-free, and disease-specific survival (all p < 0.05). The functional experiments suggested that the overexpression of LDOC1 contributed to increased G1 and G2 stages in Huh7, while increased G2 stage in Hep3B, and decreased cell proliferation, clone formation, and migration, as well as increased the apoptosis rate compared with the control group (all p < 0.05). Furthermore, LDOC1 up-regulation reduced the p-AKT/AKT and p-mTOR/mTOR, which indicates an inactivation of the AKT/mTOR pathway. Conclusion: The tumor-suppressor LDOC1 varied in HCC and non-HCC tissues, which can serve as a candidate prognostic biomarker. LDOC1 influenced survival by affecting proliferation, colony formation, cell cycle, apoptosis, and migration ability, which might be attributed to the AKT/mTOR inhibition in HCC.

VIPR1 promoter methylation promotes transcription factor AP-2α binding to inhibit VIPR1 expression and promote hepatocellular carcinoma cell growth in vitro

To explore the transcriptional regulation mechanism and biological function of low expression of vasoactive intestinal peptide receptor 1 (VIPR1) in hepatocellular carcinoma (HCC). We constructed plasmids carrying wild-type VIPR1 promoter or two mutant VIPR1 promoter sequences for transfection of the HCC cell lines Hep3B and Huh7, and examined the effect of AP-2α expression on VIPR1 promoter activity using dual-luciferase reporter assay. Pyrosequencing was performed to detect the changes in VIPR1 promoter methylation level in HCC cells treated with a DNA methyltransferase inhibitor (DAC). Chromatin immunoprecipitation was used to evaluate the binding ability of AP-2α to VIPR1 promoter. Western blotting was used to assess the effect of AP-2α knockdown on VIPR1 expression and examine the differential expression of VIPR1 in the two cell lines. The effects of VIPR1 overexpression and knockdown on the proliferation, cell cycle and apoptosis of HCC cells were analyzed using CCK8 assay and flow cytometry. We also observed the growth of HCC xenograft with lentivirus-mediated over-expression of VIPR1 in nude mice. Compared with the wild-type VIPR1 promoter group, co-transfection with the vector carrying two promoter mutations and the AP-2α-over-expressing plasmid obviously restored the luciferase activity in HCC cells (P < 0.05). DAC treatment of the cells significantly decreased the methylation level of VIPR1 promoter and inhibited the binding of AP-2α to VIPR1 promoter (P < 0.01). The HCC cells with AP-2α knockdown showed increased VIPR1 expression, which was lower in Huh7 cells than in Hep3B cells. VIPR1 overexpression in HCC cells caused significant cell cycle arrest in G2/M phase (P < 0.01), promoted cell apoptosis (P < 0.001), and inhibited cell proliferation (P < 0.001), while VIPR1 knockdown produced the opposite effects. In the tumor-bearing nude mice, VIPR1 overexpression in the HCC cells significantly suppressed the increase of tumor volume (P < 0.001) and weight (P < 0.05). VIPR1 promoter methylation in HCC promotes the binding of AP-2α and inhibits VIPR1 expression, while VIPR1 overexpression causes cell cycle arrest, promotes cell apoptosis, and inhibits cell proliferation and tumor growth.

Circular dorsal ruffles disturb the growth factor-induced PI3K-AKT pathway in hepatocellular carcinoma Hep3B cells

BackgroundCircular dorsal ruffles (CDRs) are rounded membrane ruffles induced on the dorsal surfaces of cells stimulated by growth factors (GF). They can serve as signal platforms to activate AKT protein kinase. After GF stimulation, phosphatidylinositol 3-kinase (PI3K) generates phosphatidylinositol (3,4,5)-triphosphate (PIP3) in the plasma membrane. PIP3 accumulates inside CDRs, recruits AKT into the structures, and phosphorylates them (pAKT). Given the importance of the PI3K-AKT pathway in GF signaling, CDRs are likely involved in cell growth. Interestingly, some cancer cell lines express CDRs. We hypothesized that CDRs contribute to carcinogenesis by modulating the AKT pathway. In the present study, we identified CDR-expressing cancer cell lines and investigated their cellular functions.MethodsCDR formation was examined in six cancer cell lines in response to epidermal growth factor (EGF) and insulin. The morphology of the CDRs was characterized, and the related signaling molecules were observed using confocal and scanning electron microscopy. The role of CDRs in the AKT pathway was studied using biochemical analysis. The actin inhibitor cytochalasin D (Cyto D) and the PI3K inhibitor TGX221 were used to block CDRs.ResultsGF treatment induced CDRs in the hepatocellular carcinoma (HCC) Hep3B cell line, but not in others, including HCC cell lines HepG2 and Huh7, and the LO2 hepatocyte cell line. Confocal microscopy and western blot analysis showed that the PI3K-PIP3-AKT pathway was activated at the CDRs and that receptor proteins were recruited to the structures. Cyto D and TGX221 completely blocked CDRs and partially attenuated GF-induced pAKT. These results indicate that CDRs regulate the receptor-mediated PI3K-AKT pathway in Hep3B cells and the existence of CDR-independent pAKT mechanisms.ConclusionsOur results showed that CDRs modulate the AKT pathway in Hep3B cells. Since CDRs were not observed in other HCC and hepatocyte cell lines, we propose that CDRs in Hep3B would determine the carcinoma characteristic of the cell by aberrantly triggering the AKT pathway. Signaling molecules involved in CDR formation are promising therapeutic targets for some types of HCC.9eb9aTWDk6mAQeLNgQGeJaVideo abstract

Goji Ferment Ameliorated Acetaminophen-Induced Liver Injury in vitro and in vivo.

This study aimed to investigate the hepatoprotective effects of lyophilized powder of goji ferment (LPGF) against acetaminophen (APAP)-induced hepatic damage in Hep3B cells and in mice. Eleven strains of lactic acid bacteria (LAB) were selected and their hepatoprotection against APAP-induced cellular damage in Hep3B cell line was evaluated. Four strains of LAB, including BCRC11652 (Leuconostoc mesenteroides subsp. mesenteroides), BCRC14619 (Lactobacillus gasseri), KODA-1 (Pediococcus acidilactici), and KODA-2 (Limosilactobacillus fermentum), have hepatoprotective potential against APAP in vitro. Goji significantly stimulated the growth of individual and combined strains of LAB and the optimal fermented condition was the treatment of goji at 10% (w/w) for 24h. The prepared lyophilized powder of goji ferment (LPGF) containing fifteen combinations of LAB strains was used to explore their hepatoprotection in vitro. LPGF containing all combinations of LAB strains, except for KODA-2, significantly restored APAP-reduced cell viability and improved APAP-increased cellular levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). In mice model, LPGF containing BCRC11652, BCRC14619, and KODA-2 was chosen to evaluate its hepatoprotection against APAP-induced liver injury. LPGF at diverse doses have a tendency but no significant improvement on APAP-reduced body weight gain and liver weight. LPGF significantly decreased APAP-increased serum ALT and AST levels in a dose-dependent manner. At the end of experiment, LPGF significantly and dose-dependently reversed APAP-reduced activities of GSH and antioxidant enzymes, including glutathione peroxidase, superoxide dismutase, and catalase in hepatic tissue. Overall, LPGF was demonstrated to exhibit hepatoprotection against APAP-induced liver injury in vitro and in vivo.

Second Generation Small Molecule Inhibitors of Gankyrin for the Treatment of Pediatric Liver Cancer.

Simple SummaryNew therapy options are needed for children with liver cancer. The goal of this study was to evaluate the role of three new compounds in the treatment of liver cancer cells. These compounds all inhibit a protein called Gankyrin, which is known to promote liver cancer by destroying tumor suppressor proteins. We demonstrated that liver cancer cells have significantly reduced proliferation when treated with these compounds by preventing the degradation of tumor suppressor proteins. We also discovered that these compounds enhance the effects of doxorubicin, which is a chemotherapy drug commonly used in liver cancer. These results support continued efforts in Gankyrin-based therapy for the treatment of pediatric liver cancer.Background: Gankyrin, a member of the 26S proteasome, is an overexpressed oncoprotein in hepatoblastoma (HBL) and hepatocellular carcinoma (HCC). Cjoc42 was the first small molecule inhibitor of Gankyrin developed; however, the IC50 values of >50 μM made them unattractive for clinical use. Second-generation inhibitors demonstrate a stronger affinity toward Gankyrin and increased cytotoxicity. The aim of this study was to characterize the in vitro effects of three cjoc42 derivatives. Methods: Experiments were performed on the HepG2 (HBL) and Hep3B (pediatric HCC) cell lines. We evaluated the expression of TSPs, cell cycle markers, and stem cell markers by Western blotting and/or real-time quantitative reverse transcription PCR. We also performed apoptotic, synergy, and methylation assays. Results: The treatment with cjoc42 derivatives led to an increase in TSPs and a dose-dependent decrease in the stem cell phenotype in both cell lines. An increase in apoptosis was only seen with AFM-1 and -2 in Hep3B cells. Drug synergy was seen with doxorubicin, and antagonism was seen with cisplatin. In the presence of cjoc42 derivatives, the 20S subunit of the 26S proteasome was more available to transport doxorubicin to the nucleus, leading to synergy. Conclusion: Small-molecule inhibitors for Gankyrin are a promising therapeutic strategy, especially in combination with doxorubicin.

Design, synthesis, and biological evaluation of a new series of pyrazole derivatives: Discovery of potent and selective JNK3 kinase inhibitors

The design, synthesis, and biological activities of a new series of pyrazole derivatives are reported. The target compounds 1a-1w were initially investigated against NCI-60 cancer cell lines. Compounds 1f, 1h, 1k, and 1v exerted the highest anti-proliferative activity over the studied panel of cancer cell lines. Compound 1f showed the most potent activity, and it is more potent than sorafenib in 29 cancer cell lines of different types and more potent than SP600125 against almost all the tested cancer cell lines. It also exerted sub-micromolar IC50 values (0.54–0.98 µM) against nine cell lines. Moreover, the 23 target compounds were tested against Hep3B and HepG2 hepatocellular carcinoma cell lines, of which compounds 1b, 1c, and 1h showed the strongest anti-proliferative activity. The most potent anticancer compounds (1b, 1c, 1f, and 1h) demonstrated a high selectivity towards cancer cells vis-à-vis normal cells. Compounds1f and 1h induced apoptosis and mild necrosis upon testing against RPMI-8226 leukemia cells. Kinase profiling of this series led to the discovery of two potent and selective JNK3 inhibitors, compounds 1c and 1f with an IC50 values of 99.0 and 97.4 nM, respectively. Both compounds showed a good inhibitory effect against JNK3 kinase in the whole-cell NanoBRET assay. This finding was further supported through molecular modeling studies with the JNK3 binding site. Moreover, compounds 1c and 1f demonstrated a very weak activity against CYP 2D6, CYP 3A4, and hERG ion channels.

Abstract 2752: Preclinical characterization of FLEX-NK࣪ tetravalent NKp46 engager directed against GPC3 (CYT-303) alone or in combination with iPSC derived Natural Killer cells (iNKs) against hepatocellular carcinoma (HCC)

Abstract Glypican-3 (GPC3) is highly expressed in multiple solid tumors including HCC while it is hardly expressed in adult normal tissues except placenta. GPC3 promotes Wnt-dependent cell proliferation, and its expression is correlated with poor prognosis in HCC. NK cells exhibit innate anti-tumor activity owing to the expression of multiple activating receptors, such as NKp46. NKp46 is expressed in all NK cells including tumor-infiltrating NK cells. FLEX-NKTrademark is a proprietary platform for production of tetravalent IgG1-like multifunctional antibody NK engagers with a novel FLEX-linker to allow for simultaneous binding of both the targeted cancer cells and NK cells. A novel humanized NKp46 binder that does not induce NKp46 internalization and a humanized GPC3 binder that targets the membrane-proximal lobe of the GPC3 were combined on the novel FLEX-NKTrademark scaffold to create the NK engager CYT-303. CYT-303 has higher affinity for GPC3 compared to NKp46 to skew binding preferentially to GPC3 expressing tumor cells prior to binding NK cells expressing NKp46 to optimize targeted killing. CYT-303 showed significantly higher dose dependent peripheral blood NK cell redirected cytotoxicity and degranulation against GPC3 expressing Hep3B tumor cells compared to GPC3 or NKp46 monoclonal antibodies alone suggesting that co-engagement of NKp46 and GPC3 via an immunological synapse is required for optimal tumor killing by CYT-303. Low NK cell numbers or suppression of NK cell function in the tumor microenvironment may limit the clinical activity of FLEX-NKTrademark engagers. iNK cells derived from iPSC, a uniform starting material with unlimited self-renewal capabilities, can be expanded to produce a universal off-the-shelf allogeneic therapy that can be used in combination with FLEX-NKTrademark engagers. We studied the efficacy of the combination of a FLEX-NKTrademark antibody and iNKs. The iNK cells express high levels of multiple activation receptors including NKp46 and showed good cytotoxic activity against HCC cell line Hep3B. The iNKs also showed anti-tumor activity in NSG-hIL15 mice bearing HCC subcutaneous tumors as demonstrated by the presence of CD56+CD3-, NKp46+, NKG2D+ iNKs in the tumors at day 21 post-intratumoral injection of the iNKs. CYT-303 greatly enhanced the cytotoxic activity of iNKs and cytolysis of Hep3B tumor cells in-vitro. CYT-303 and iNK cells, alone or in combination, demonstrate anti-tumor activity against HCC that warrants clinical development. Citation Format: Antonio Arulanandam, Liang Lin, Hao-Ming Chang, David Zou, Melissa Triggiano, Nejmi Dilmac, Yinan Wang, Shira Kahlon, Stanley Frankel, Jean Kadouche, Daniel Teper, Ofer Mandelboim, Wei Li. Preclinical characterization of FLEX-NK࣪ tetravalent NKp46 engager directed against GPC3 (CYT-303) alone or in combination with iPSC derived Natural Killer cells (iNKs) against hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2752.

Engineered, fully human nanobody-based CAR T cells have enhanced antitumor activity against hepatocellular carcinoma in preclinical models.

e14512 Background: Glypican 3 (GPC3), a cell surface protein associated with early development, is an established biomarker of hepatocellular carcinoma (HCC). In a vast compendium of work, HN3, a human GPC3 nanobody (VH) isolated from a phage library, was shown to abrogate HCC tumors and block the distally located Wnt binding domain of GPC3. To date, it remains challenging how to precisely engineer CAR T cells for improved efficacy in solid tumors. In the context of hepatocellular carcinoma, we determined how hinges and transmembrane portions of varying structures and sizes affect CAR T cell function. Here, we show how rationally designed, engineered Chimeric Antigen Receptor (CAR) T cells containing HN3, demonstrate enhanced CAR T activity in HCC. Methods: We generated and compared multiple permutations of GPC3 targeted CAR T cells containing HN3, CD8, CD28, IgG4 and Fc domains. Incorporation of HN3, into multiple CAR formats yielded nine constructs, which were screened for antigen-specific and antigen-independent signaling using a luciferase reporter system in Hep3B, Huh7 and HepG2 cell lines. For in vivo assessment, immunodeficient NSG mice were intraperitonially injected with Hep3B or Huh7 cells and treated with a single dose (5 or 15 million) of engineered HN3 CAR T cells. Blood was collected at 2, 4 and 5 weeks to assess proliferation, T cell subtypes and exhaustion. Results: In vitro, the HN3-IgG4H-CD28TM CAR T cells induced high specificity and cytotoxic activity. HN3-IgG4H-CD28TM CAR T cells markedly improved HN3 cell killing activity by 30-40% in low (1.6:1) and high (25:1) effector to target ratios. Similarly, in vivo, in a high antigen environment, two determinative changes in the hinge region and transmembrane domains led to complete tumor eradication in immunodeficient mice bearing HCC tumors within 7-10 days. Moreover, HN3-IgG4H-CD28TM CAR T cells maintained specific tumor killing and averted exhaustion by producing a clear T cell response signature of enriched cytotoxic-memory (Temra) CD8+ along with a subset of naïve T cells. Conclusions: Engineered nanobody based CAR T cells containing the appropriate hinge and transmembrane domains can lead to determinative T cell signaling capable of inducing swift and durable eradication of HCC tumors. Altogether, we show that engineered HN3 CAR T cell therapy demonstrates strong potential for treatment of aggressive HCCs enabled by Wnt dysregulation.

Synthesis and Studies of Anticancer and Antimicrobial Activity of New Phenylurenyl Chalcone Derivatives

Background: Phenylurenyl chalcone structures have the potential to act as a scaffold in anticancer drug discovery. Methods: N-Phenethyl-N'-{4-[(2E)-3-phenylprop-2-enoyl]phenyl}urea, 4/3-[(2E)-3-substitutedphenylprop-2-enoyl]phenyl}-N-phenylurea,4/3-[(2E)-3-substitutedphenyl prop-2-enoyl]phenyl}-N-methylphenyl urea and {4/3-[(2E)-3-substitutedphenylprop-2-enoyl]phenyl}-N-ethylphenyl urea derivatives(1-35)were prepared and evaluated for their anticancer and antimicrobial activity against A-549 Hep-3B, HT-29, CF-7, PC-3, K-562 NIH-3T3 and Huh-7 cell lines and against Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739) and Candida albicans (ATCC 10231), respectively. Results: While compounds 2, 26, 29, and 34 showed moderate cytotoxic activity on cell line Huh 7, compounds 14 (IC50: 6.42 µM), 16 (IC50: 5.64 µM), 19 (IC50: 6.95 µM) and 34 (IC50: 6.87 µM) showed good cytotoxic activity on Huh-7 cell line close to Sorafenib (IC50: 4.29 µM) (as reference). MIC values of compounds 4 and 22 against E. coli were 25 μg/ml, of compounds 3, 14 and 29 against P. aeruginosa 25 μg/ml and of compounds 11 and 33 against S. aureus 25 μg/ml. On the other hand, the minimum inhibitory concentration of all tested compounds against C. albicans was 25 μg/ml. Conclusion: N-Phenethyl-N'-{4-[(2E)-3-phenylprop-2-enoyl]phenyl}urea may be a new candidate to be developed as an anticancer compound.

Sphk1 and Sphk2 Differentially Regulate Erythropoietin Synthesis in Mouse Renal Interstitial Fibroblast-like Cells.

Erythropoietin (Epo) is a crucial hormone regulating red blood cell number and consequently the hematocrit. Epo is mainly produced in the kidney by interstitial fibroblast-like cells. Previously, we have shown that in cultures of the immortalized mouse renal fibroblast-like cell line FAIK F3-5, sphingosine 1-phosphate (S1P), by activating S1P1 and S1P3 receptors, can stabilize hypoxia-inducible factor (HIF)-2α and upregulate Epo mRNA and protein synthesis. In this study, we have addressed the role of intracellular iS1P derived from sphingosine kinases (Sphk) 1 and 2 on Epo synthesis in F3-5 cells and in mouse primary cultures of renal fibroblasts. We show that stable knockdown of Sphk2 in F3-5 cells increases HIF-2α protein and Epo mRNA and protein levels, while Sphk1 knockdown leads to a reduction of hypoxia-stimulated HIF-2α and Epo protein. A similar effect was obtained using primary cultures of renal fibroblasts isolated from wildtype mice, Sphk1−/−, or Sphk2−/− mice. Furthermore, selective Sphk2 inhibitors mimicked the effect of genetic Sphk2 depletion and also upregulated HIF-2α and Epo protein levels. The combined blockade of Sphk1 and Sphk2, using Sphk2−/− renal fibroblasts treated with the Sphk1 inhibitor PF543, resulted in reduced HIF-2α and Epo compared to the untreated Sphk2−/− cells. Exogenous sphingosine (Sph) enhanced HIF-2α and Epo, and this was abolished by the combined treatment with the selective S1P1 and S1P3 antagonists NIBR-0213 and TY52156, suggesting that Sph was taken up by cells and converted to iS1P and exported to then act in an autocrine manner through S1P1 and S1P3. The upregulation of HIF-2α and Epo synthesis by Sphk2 knockdown was confirmed in the human hepatoma cell line Hep3B, which is well-established to upregulate Epo production under hypoxia. In summary, these data show that sphingolipids have diverse effects on Epo synthesis. While accumulation of intracellular Sph reduces Epo synthesis, iS1P will be exported to act through S1P1+3 to enhance Epo synthesis. Furthermore, these data suggest that selective inhibition of Sphk2 is an attractive new option to enhance Epo synthesis and thereby to reduce anemia development in chronic kidney disease.

Design synthesis of Y-90 glass microspheres and study of their therapeutic effects on mouse liver cancer cell line Hep3B

In this article, a system for synthesizing Y-90 glass microspheres (Y-90-GM) was successfully designed in the Da Lat nuclear reactor (Vietnam), and the therapeutic effects of Y-90-GM on mice liver cancer cell line Hep3B were studied. The effects of synthesis factors, including heating time, heating temperature, gas flow rate, sample conduit length and diameter, were investigated to establish the optimal parameters. The size and shape of Y-90-GM were checked by field emission scanning electron microscope, and the radioactivity measurement was performed on a dosimeter. The results indicated that the optimal conditions for the synthesis of Y-90-GM were determined as the heating temperature of 1600 °C, heating time of 2 h, conduit length and diameter of 50 cm and 3.6 cm, and gas/oxygen flow rate of 15 mph. The Y-90-GM samples obtained at the optimal parameters have a size of 18–30 μm with a density of 3.53 g cm−3 and a specific radioactivity of 630 mCi g−1. The results of the therapeutic study on mice liver cancer cell line Hep3B showed that after two weeks of treatment with Y-90-GM (1mCi/mouse), the tumor volume was reduced by about 30.7% and after 3 consecutive treatment cycles, the liver cancer tumor was completely reduced. It was demonstrated that Y-90-GM is promising radiopharmaceuticals in the treatment of liver cancer by the radioembolization method.

LncRNA SLC16A1-AS1 contributes to the progression of hepatocellular carcinoma cells by modulating miR-411/MITD1 axis.

BackgroundHepatocellular carcinoma (HCC) is a common malignancy with high morbidity. The current study aimed to explore the molecular mechanism of lncRNA SLC16A1‐AS1 in the tumorigenesis of HCC.Material and methodsThe expression of SLC16A1‐AS1 and miR‐411 was examined in clinical HCC tissues. HCC cell lines Hep3B and Huh‐7 were employed and transfected with si‐SLC16A1‐AS1. The correlation between SLC16A1‐AS1 and miR‐411 was verified by luciferase reporter assay. Cell viability was detected by CCK‐8 assay. Cell migration and invasion capacity were examined by transwell assay. The protein level of MITD1 was analyzed by western blotting.ResultsThe expression of SLC16A1‐AS1 markedly increased in HCC tissues and cell lines. Subsequent studies identified SLC16A1‐AS1 as a downstream target of miR‐411. In addition, SLC16A1‐AS1 knockdown and miR‐411 overexpression significantly stagnated the progression of HCC cells. SLC16A1‐AS1 knockdown also downregulated MITD1 levels.ConclusionOur findings showed that SLC16A1‐AS1 was overexpressed in HCC cells and tissues. SLC16A1‐AS1 promoted the malignant characteristics of HCC cells and acted as an oncogene. Its regulatory effect may be associated with miR‐411/MITD1 axis. Therefore, SLC16A1‐AS1 has the potential to be used as a biomarker or therapeutic target for the treatment of HCC.

High PPT1 expression predicts poor clinical outcome and PPT1 inhibitor DC661 enhances sorafenib sensitivity in hepatocellular carcinoma

BackgroundAdaptive resistance and side effects of sorafenib treatment result in unsatisfied survival of patients with hepatocellular carcinoma (HCC). Palmitoyl-protein thioesterase 1 (PPT1) plays a critical role in progression of various cancers. However, its role on prognosis and immune infiltrates in HCC remains unclarified.MethodsBy data mining in the Cancer Genome Atlas databases, the role of PPT1 in HCC were initially investigated. Furthermore, HCC cell lines Hep 3B and Hep 1-6 were treated with DC661 or siRNA against PPT1. The biological function of PPT1 was determined by CCK-8 test, colony formation assay, TUNEL staining, immunofluorescence staining, Western blot test, and PI-Annexin V apoptosis assays in vitro. Animal models of subcutaneous injection were applied to investigate the therapeutic role of targeting PPT1.ResultsWe found that PPT1 levels were significantly upregulated in HCC tissues compared with normal tissues and were significantly associated with a poor prognosis. Multivariate analysis further confirmed that high expression of PPT1 was an independent risk factor for poor overall survival of HCC patients. We initially found that PPT1 was significantly upregulated in sorafenib-resistant cell lines established in this study. Upon sorafenib treatment, HCC cells acquired adaptive resistance by inducing autophagy. We found that DC661, a selective and potent small-molecule PPT1-inhibitor, induced lysosomal membrane permeability, caused lysosomal deacidification, inhibited autophagy and enhanced sorafenib sensitivity in HCC cells. Interestingly, this sensitization effect was also mediated by the induction mitochondrial pathway apoptosis. In addition, the expression level of PPT1 was associated with the immune infiltration in the HCC tumor microenvironment, and PPT1 inhibitor DC661 significantly enhanced the anti-tumor immune response by promoting dendritic cell maturation and further promoting CD8+ T cell activation. Moreover, DC661 combined with sorafenib was also very effective at treating tumor models in immunized mice.ConclusionsOur findings suggest that targeting PPT1 with DC661 in combination with sorafenib might be a novel and effective alternative therapeutic strategy for HCC.

Guided isolation of daphnane-type diterpenes from Daphne genkwa by molecular network strategies

A molecular networking-guided study on the Daphne genkwa Sieb. et Zucc led to the isolation of twelve daphnane-type diterpenoids including four undescribed compounds, yuanhuakines A–D. Their structures were elucidated by spectroscopic analyses, ECD calculations, and single-crystal X-ray diffraction analysis. All isolates were evaluated for their inhibitory activity against the A549, Hep3B, and MCF-7 cell lines. The majority of compounds inhibited A549 cells with IC50 values ranging from 7.77 to 20.56 μM, and their structure-activity relationship is preliminarily discussed. Five of these compounds were selected for further experiments, and they appear to inhibit A549 cell lines by inducing apoptosis.

Evaluation of the expression of fatty acid synthase and O-GlcNAc transferase in patients with liver cancer by exploration of transcriptome databases and experimental approaches.

Tumor occurrence and development are closely related to metabolism abnormalities. One of the metabolic networks that is dysregulated during carcinogenesis is the fatty acid synthesis pathway, which is mainly controlled by fatty acid synthase (FASN). We previously demonstrated in proliferating HepG2 liver cancer cells that FASN expression depends on the catalytic activity of O-GlcNAc transferase (OGT) and the activation of the mechanistic/mammalian target of rapamycin (mTOR) pathway. The aim of the present study was to go further in these investigations by analyzing datasets and tissues of patients with liver cancer. To that purpose, transcriptome databases were explored, and reverse transcription-quantitative PCR, western blotting and immunohistochemistry were used. Database analyses revealed that FASN and OGT gene expression was higher in certain cancer tissues, including liver hepatocellular carcinoma, compared with that in non-cancerous tissues. At the protein level, FASN expression was higher in the liver cancer-derived cell lines HepG2 and Hep3B compared with the immortalized human hepatocytes IHH cell line. However, neither the expression of OGT nor of its product O-GlcNAcylation showed any significant difference among the three hepatic cell lines. Subsequently, the expression of FASN and OGT at the protein and mRNA levels was evaluated in human liver cancer and non-tumoral tissues from the same patients with different liver lesions. The results from western blotting demonstrated a significant increase in OGT ands O-GlcNAcylation expression in liver cancer tissues independently of the type of lesion characterizing the non-tumoral counterpart. As previously reported for HepG2 proliferating cells, the protein level of FASN was positively correlated with the activation of mTOR and, although a rather upward trend, a high variability in its expression was monitored between patients. However, the results from immunohistochemistry showed no particular modification for OGT and O-GlcNAcylation expression and a significant increase in FASN expression in cancer tissues compared with that in adjacent non-tumoral tissues. Non-significant changes were observed for FASN and OGT mRNA levels between tumoral and non-tumoral samples, with a high variability between patients. Taken together, these results demonstrated that FASN expression was higher in hepatic cancer tissues in comparison with non-tumoral tissues. Furthermore, OGT expression and activity were shown to vary greatly between cell or cancer type, making any generalization difficult.