Hepatocellular Carcinoma 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.

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.

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.

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.

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.

CHEMOSENSITIZING EFFECTS OF GDF11 IN HUMAN HEPATOCELLULAR CARCINOMA CELLS

Hepatocellular carcinoma (HCC) ranks as the second leading cause of cancer death globally; this neoplasm accounts for approximately 90% of liver cancers, and about 850,000 new cases are reported annually. Several factors increase the likelihood of developing HCC, such as excessive alcohol consumption, hepatitis B and C virus infection, metabolic syndrome, and a diet high in lipids and cholesterol. The chronic lesions that the liver can suffer due to the aggression of these factors usually generate lower grade pathologies such as fatty liver, hepatitis, and cirrhosis, which can evolve into HCC. In 2019 Gerardo-Ramirez and collaborators from our group reported the ability of GDF11 to subtract aggressiveness to several HCC-derived cell lines HCC (Huh7, Hep3B, SNU-182, Hepa 1-6 and HepG2); they found that GDF11 reduced proliferation, metastatic capacity, colony, and spheroid formation and invasiveness in those cell lines. Findings by Gerardo-Ramirez et al. (2019) identified transcriptional repression of cyclins D1 and A and overexpression of p27. Additionally, an increase in the expression of epithelial markers E-cadherin and Occludin was observed; conversely, mesenchymal-type features such as N-cadherin and Snail decreased with GDF11 treatment, confirming that this growth factor-induced a mesenchymal to epithelial transition. Furthermore, our group reported the effect of GDF11 in reducing lipid content, especially cholesterol and triglycerides. It was also confirmed that GDF11 reduced mevalonate pathway proteins in Huh7 and Hep3B liver cancer cell lines. Additionally, they reported that GDF11 was able to impair mitochondrial functionality and its structure. Moreover, GDF11 treatment induced an alteration of glycolytic capacity and oxygen consumption rate in these models. To determine the sensitizing effect of GDF11 in the Hep3B cell line. Specific: To determine the capacity of GDF11 in the reduction of the EC50 of cisplatin. We used the HCC cell line Hep3B (ATCC). A 72-h pretreatment with GDF11 or without was performed; then we treated the cells with cisplatin at various concentrations (0, 2.5, 5, 10, 15, 15, 25, 50, and 100 μM), incubated for 48 h, and cell viability assay was performed by crystal violet. In our experiments, GDF11 has shown an increased sensitivity of Hep3B cells to cisplatin treatment by significantly reducing the mean effective dose (EC50) from 22.26 μM to 8.11 μM this result was observed by crystal violet assay and by light microscopy. Results demonstrate that GDF11 has sensitizing effects against cisplatin treatment on the liver tumor cell line Hep3B. This agrees with previous results of our group where a detrimental impact in liver tumor cells is observed by the GDF11 treatment and contrast with other works where TGF-β family members have chemoresistance effects. GDF11 pretreatment sensitizes the HCC cell line Hep3B by reducing the cisplatin EC50. Conacyt Fronteras de la Ciencia 1320 The authors declare that there is no conflict of interest.

Murine double minute gene 2 (MDM2) promoted hepatocellular carcinoma (HCC) cell growth by targeting fructose-1,6-bisphosphatase (FBP1) for degradation

To study the roles and association of murine double minute gene 2 (MDM2) and fructose-1,6-biphosphatase (FBP1) in human hepatocellular carcinoma (HCC), growth response of human HCC cells was assessed using proliferation and apoptosis assay. Pro-survival AKT signaling associated proteins (p-AKT, survivin and cleaved caspase 3) were assessed using western blotting. The correlation between MDM2 and FBP1 was assessed using co-immunoprecipitation combined with ubiquitination assay. Our data suggested that low expression of FBP1 was correlated with high levels of MDM2 in HCC cell lines (Huh7 and Hep3B). Overexpression of FBP1 resulted in anti-proliferation, pro-apoptosis, the up-regulation of cleaved caspase 3 while the downregulation of survivin and phosphor (p)-AKT, however, knockdown of FBP1 led to the opposite. Furthermore, overexpression of MDM2 potently reversed FBP1-induced proliferation inhibition and apoptosis, while Nutlin-3 (an MDM2 inhibitor) reversed the change trends induced by FBP1 knockdown in the aforementioned events. Lastly, but not least importantly, our data elucidated that MDM2 binds directly to FBP1 and promotes FBP1 ubiquitination. In conclusion, our data firstly suggested the involvement of FBP1 and its association with MDM2 in HCC cell growth. MDM2-FBP1-regulated HCC cell growth and the activation of AKT were mediated, at least in part, through FBP1 degradation.

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.

CKS1B promotes the progression of hepatocellular carcinoma by activating JAK/STAT3 signal pathway

ABSTRACT Hepatocellular carcinoma (HCC) is a malignancy of considerable concern due to its continuous increase in morbidity and mortality. This study attempts to identify the molecules that play a key role in the progression of HCC, explore its potential mechanism, and provide more target choices for targeted therapy. Using overexpression plasmid and shRNA, CKS1B was respectively overexpressed and knocked down to explore its biological function roles in HCC progression and development. MTT and colony formation assays showed that knockdown of CKS1B inhibited the survival and proliferation of HCC cell lines (Hep3B and Huh7). The flow cytometry and western blot analysis showed that knockdown of CKS1B significantly induced the apoptosis of Hep3B and Huh7 cells. The wound healing and transwell invasion assays showed that knockdown of CKS1B had a significant inhibitory effect on the migration and invasion of Hep3B and Huh7 cells. These functional tests confirmed that CKS1B acts as an oncogene that regulates the malignant progression of HCC. Moreover, this study also demonstrated that knockdown of CKS1B inhibited the activation of JAK/STAT3 pathway, evidenced by the significantly downregulated p-STAT3 protein expression. Furthermore, knockdown of CKS1B also downregulated STAT3 target genes TIMP-1, Bcl-2 and VEGF, which were involved in controlling cell apoptosis and migration. On the contrary, overexpression of CKS1B caused the completely opposite results. Taken together, CKS1B acts as an oncogene to promote the proliferation and metastasis of HCC cells by activating JAK/STAT3 signaling pathway.

Cancer Stem-Like Phenotype of Mitochondria Dysfunctional Hep3B Hepatocellular Carcinoma Cell Line.

Mitochondria are major organelles that play various roles in cells, and mitochondrial dysfunction is the main cause of numerous diseases. Mitochondrial dysfunction also occurs in many cancer cells, and these changes are known to affect malignancy. The mitochondria of normal embryonic stem cells (ESCs) exist in an undifferentiated state and do not function properly. We hypothesized that mitochondrial dysfunction in cancer cells caused by the depletion of mitochondrial DNA might be similar to the mitochondrial state of ESCs. We generated mitochondria dysfunctional (ρ0) cells from the Hep3B hepatocellular carcinoma cell line and tested whether these ρ0 cells show cancer stem-like properties, such as self-renewal, chemotherapy resistance, and angiogenesis. Compared with Hep3B cells, the characteristics of each cancer stem-like cell were increased in Hep3B/ρ0 cells. The Hep3B/ρ0 cells formed a continuous and large sphere from a single cell. Additionally, the Hep3B/ρ0 cells showed resistance to the anticancer drug doxorubicin because of the increased expression of ATP-binding cassette Subfamily B Member 1. The Hep3B/ρ0 conditioned medium induced more and thicker blood vessels and increased the mobility and invasiveness of the blood vessel cells. Therefore, our data suggest that mitochondrial dysfunction can transform cancer cells into cancer stem-like cells.

Cold Atmospheric Pressure Plasma-Activated Medium Induces Selective Cell Death in Human Hepatocellular Carcinoma Cells Independently of Singlet Oxygen, Hydrogen Peroxide, Nitric Oxide and Nitrite/Nitrate.

Cold atmospheric pressure plasma (CAP) and plasma-activated medium (PAM) induce cell death in diverse cancer cells and may function as powerful anti-cancer agents. The main components responsible for the selective anti-cancer effects of CAP and PAM remain elusive. CAP or PAM induces selective cell death in hepatocellular carcinoma cell lines Hep3B and Huh7 containing populations with cancer stem cell markers. Here, we investigated the major component(s) of CAP and PAM for mediating the selective anti-proliferative effect on Hep3B and Huh7 cells. The anti-proliferative effect of CAP was mediated through the medium; however, the reactive oxygen species scavenger N-acetyl cysteine did not suppress PAM-induced cell death. Neither high concentrations of nitrite or nitrite/nitrate nor a low concentration of H2O2 present in the PAM containing sodium pyruvate affected the viability of Hep3B and Huh7 cells. Inhibitors of singlet oxygen, superoxide anions, and nitric oxide retained the capacity of PAM to induce anti-cancer effects. The anti-cancer effect was largely blocked in the PAM prepared by placing an aluminum metal mesh, but not a dielectric PVC mesh, between the plasma source and the medium. Hence, singlet oxygen, hydrogen peroxide, nitric oxide, and nitrite/nitrate are not the main factors responsible for PAM-mediated selective death in Hep3B and Huh7 cells. Other factors, such as charged particles including various ions in CAP and PAM, may induce selective anti-cancer effects in certain cancer cells.

3,3′-Diindolylmethane Suppresses the Growth of Hepatocellular Carcinoma by Regulating Its Invasion, Migration, and ER Stress-Mediated Mitochondrial Apoptosis

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death worldwide with limited treatment options. Biomarker-based active phenolic flavonoids isolated from medicinal plants might shed some light on potential therapeutics for treating HCC. 3,3′-diindolylmethane (DIM) is a unique biologically active dimer of indole-3-carbinol (I3C), a phytochemical compound derived from Brassica species of cruciferous vegetables—such as broccoli, kale, cabbage, and cauliflower. It has anti-cancer effects on various cancers such as breast cancer, prostate cancer, endometrial cancer, and colon cancer. However, the molecular mechanism of DIM involved in reducing cancer risk and/or enhancing therapy remains unknown. The aim of the present study was to evaluate anti-cancer and therapeutic effects of DIM in human hepatoma cell lines Hep3B and HuhCell proliferation was measured with MTT and trypan blue colony formation assays. Migration, invasion, and apoptosis were measured with Transwell assays and flow cytometry analyses. Reactive oxygen species (ROS) intensity and the loss in mitochondrial membrane potential of Hep3B and Huh7 cells were determined using dihydroethidium (DHE) staining and tetramethylrhodamine ethyl ester dye. Results showed that DIM significantly suppressed HCC cell growth, proliferation, migration, and invasion in a concentration-dependent manner. Furthermore, DIM treatment activated caspase-dependent apoptotic pathway and suppressed epithelial–mesenchymal transition (EMT) via ER stress and unfolded protein response (UPR). Taken together, our results suggest that DIM is a potential anticancer drug for HCC therapy by targeting ER-stress/UPR.

Apigenin Inhibits the Growth of Hepatocellular Carcinoma Cells by Affecting the Expression of microRNA Transcriptome

BackgroundApigenin, as a natural flavonoid, has low intrinsic toxicity and has potential pharmacological effects against hepatocellular carcinoma (HCC). However, the molecular mechanisms involving microRNAs (miRNAs) and their target genes regulated by apigenin in the treatment of HCC have not been addressed.ObjectiveIn this study, the molecular mechanisms of apigenin involved in the prevention and treatment of HCC were explored in vivo and in vitro using miRNA transcriptomic sequencing to determine the basis for the clinical applications of apigenin in the treatment of HCC.MethodsThe effects of apigenin on the proliferation, cell cycle progression, apoptosis, and invasion of human hepatoma cell line Huh7 and Hep3B were studied in vitro, and the effects on the tumorigenicity of Huh7 cells were assessed in vivo. Then, a differential expression analysis of miRNAs regulated by apigenin in Huh7 cells was performed using next-generation RNA sequencing and further validated by qRT-PCR. The potential genes targeted by the differentially expressed miRNAs were identified using a curated miRTarBase miRNA database and their molecular functions were predicted using Gene Ontology and KEGG signaling pathway analysis.ResultsCompared with the control treatment group, apigenin significantly inhibited Huh7 cell proliferation, cell cycle, colony formation, and cell invasion in a concentration-dependent manner. Moreover, apigenin reduced tumor growth, promoted tumor cell necrosis, reduced the expression of Ki67, and increased the expression of Bax and Bcl-2 in the xenograft tumors of Huh7 cells. Bioinformatics analysis of the miRNA transcriptome showed that hsa-miR-24, hsa-miR-6769b-3p, hsa-miR-6836-3p, hsa-miR-199a-3p, hsa-miR-663a, hsa-miR-4739, hsa-miR-6892-3p, hsa-miR-7107-5p, hsa-miR-1273g-3p, hsa-miR-1343, and hsa-miR-6089 were the most significantly up-regulated miRNAs, and their key gene targets were MAPK1, PIK3CD, HRAS, CCND1, CDKN1A, E2F2, etc. The core regulatory pathways of the up-regulated miRNAs were associated with the hepatocellular carcinoma pathway. The down-regulated miRNAs were hsa-miR-181a-5p and hsa-miR-148a-3p, and the key target genes were MAPK1, HRAS, STAT3, FOS, BCL2, SMAD2, PPP3CA, IFNG, MET, and VAV2, with the core regulatory pathways identified as proteoglycans in cancer pathway.ConclusionApigenin can inhibit the growth of HCC cells, which may be mediated by up-regulation or down-regulation of miRNA molecules and their related target genes.

Anti-tumor Effect of Oleic Acid in Hepatocellular Carcinoma Cell Lines via Autophagy Reduction.

Oleic acid (OA) is a component of the olive oil. Beneficial health effects of olive oil are well-known, such as protection against liver steatosis and against some cancer types. In the present study, we focused on OA effects in hepatocellular carcinoma (HCC), investigating responses to OA treatment (50–300 μM) in HCC cell lines (Hep3B and Huh7.5) and in a healthy liver-derived human cell line (THLE-2). Upon OA administration higher lipid accumulation, perilipin-2 increase, and autophagy reduction were observed in HCC cells as compared to healthy cells. OA in the presence of 10% FBS significantly reduced viability of HCC cell lines at 300 μM through Alamar Blue staining evaluation, and reduced cyclin D1 expression in a dose-dependent manner while it was ineffective on healthy hepatocytes. Furthermore, OA increased cell death by about 30%, inducing apoptosis and necrosis in HCC cells but not in healthy hepatocytes at 300 μM dosage. Moreover, OA induced senescence in Hep3B, reduced P-ERK in both HCC cell lines and significantly inhibited the antiapoptotic proteins c-Flip and Bcl-2 in HCC cells but not in healthy hepatocytes. All these results led us to conclude that different cell death processes occur in these two HCC cell lines upon OA treatment. Furthermore, 300 μM OA significantly reduced the migration and invasion of both HCC cell lines, while it has no effects on healthy cells. Finally, we investigated autophagy role in OA-dependent effects by using the autophagy inducer torin-1. Combined OA/torin-1 treatment reduced lipid accumulation and cell death as compared to single OA treatment. We therefore concluded that OA effects in HCC cells lines are, at least, in part dependent on OA-induced autophagy reduction. In conclusion, we report for the first time an autophagy dependent relevant anti-cancer effect of OA in human hepatocellular carcinoma cell lines.

Fraxetin Suppresses Cell Proliferation and Induces Apoptosis through Mitochondria Dysfunction in Human Hepatocellular Carcinoma Cell Lines Huh7 and Hep3B.

Fraxetin is a coumarin scaffold compound extracted from Fraxinus rhynchophylla. It has antioxidant, anti-inflammatory, hepatoprotective, and antifibrotic effects. Furthermore, fraxetin has anticancer effects in breast and lung cancer. We aimed to evaluate whether fraxetin has anticancer activity in hepatocellular carcinoma (HCC) cells and its underlying mechanism. We demonstrated the anticancer effects of fraxetin in the HCC cell lines Huh7 and Hep3B. We confirmed that fraxetin inhibited cell proliferation (42% ± 10% Huh7; 52% ± 7% Hep3B) by arresting the cell cycle and inducing apoptosis in both cell lines. Moreover, fraxetin increased reactive oxygen species production (221% ± 55% Huh7; 460% ± 73% Hep3B), depolarized the mitochondrial membranes (ΔΨm) (345% ± 160% Huh7; 462% ± 140% Hep3B), and disrupted calcium homeostasis in both HCC cell lines. Chelating calcium ions with BAPTA-AM restored proliferation in fraxetin-treated Huh7 cells but not in Hep3B cells. Fraxetin did not affect the phosphorylation of extracellular-signal-regulated kinase 1/2, whereas it decreased JNK and phosphoinositide 3-kinase signaling. Furthermore, fraxetin and mitogen-activated protein kinase pharmacological inhibitors had synergistic antiproliferative effects on HCC cells. Although our study was limited to in vitro data that require validation, we suggest that fraxetin is a potential therapeutic agent against HCC progression.

Immune checkpoint molecules are regulated by transforming growth factor (TGF)-β1-induced epithelial-to-mesenchymal transition in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with a high mortality rate. Epithelial-to-mesenchymal transition (EMT) confers cancer cells with immune evasive ability by modulating the expression of immune checkpoints in many cancers. Thus, the aim of our study is to examine the interplay between EMT and immune checkpoint molecules in HCC. A reversible EMT model was utilised with transforming growth factor (TGF)-β1 as an EMT inducer for HCC cell lines Hep3B and PLC/PRF/5. HCC cells were treated with TGF-β1 for 72 h and the EMT status and immune checkpoint expression were examined. In addition, the migratory ability of HCC cells were examined using wound healing and transwell migration assays in the reversible EMT model. siRNA-mediated knockdown of immune checkpoint molecule, B7-H3, was further utilised to validate the association between TGF-β1-mediated EMT and immune checkpoint expression in HCC. In addition, a web-based platform, SurvExpress, was utilised to evaluate the association between expression of TGF-β1 in combination with immune checkpoint molecules and overall survival in HCC patients. We observed induction of EMT upon treatment of HCC cells with TGF-β1 revealed by reduced expression of epithelial markers along with increased expression of mesenchymal markers. Withdrawal of TGF-β1 reversed the process of EMT with elevated expression of epithelial markers and reduced expression of mesenchymal markers. TGF-β1 treatment elevated the migratory potential of HCC cells which was reversed following reversal assay. Notably, during TGF-β1-induced EMT, there was upregulation of immune checkpoint molecules PD-L1 and B7-H3. However, the reversal of EMT decreased the expression of PD-L1 and B7-H3. In addition, TGF-β1 driven EMT was reversed following knockdown of B7-H3 in both HCC cells further validating the interplay between TGF-β1-mediated EMT and immune checkpoint expression in HCC. Furthermore, the coordinate expression of TGF-β1 with PD-L1 (p=0.01487) and B7-H3 (p=0.009687) was correlated with poor overall survival in 422 HCC patients. Our study has demonstrated a close association between TGF-β1-mediated EMT and regulation of immune checkpoints in HCC.

Fluorescent styrylpyrylium probes for the imaging of mitochondria in live cells.

Eight styrylpyrylium tetrafluoroborate salts have been synthesized and fully optically characterized by UV-vis absorption and fluorescence steady-state/time-resolved spectroscopies. The new dyes exhibit strong emission bands with yellow-orange colours, depending on the substituents present in the structure. Notably, the Stokes shift recorded for some of them exceeds 100 nm, a very valuable feature for biological imaging. Four of them have been assayed as biological imaging agents by confocal laser scanning microscopy (CLSM) in the human hepatoma cell line Hep3B. It has been found that all the compounds efficiently stain intracellular structures which have been identified as mitochondria through colocalization assays with MitoView (a well-known mitochondrial marker) and using carbonyl cyanide m-chlorophenyl hydrazone (CCCP) as a mitochondrial membrane potential uncoupler. Additionally, the potential ability of the studied dyes as cytotoxic drugs has been explored. The inhibitory concentration (IC50) against Hep3B was found to be in the range of 4.2 μM-11.5 μM, similar to other described anticancer drugs for the same hepatoma cell line. The combined features of a good imaging agent and potential anticancer drug make the family of the studied pyrylium salts good candidates for further theranostic studies. Remarkably, despite the extensive use of pyrylium dyes in several scientific areas (from photocatalysis to optics), there is no precedent description of a styrylpyrylium salt with potential theranostic applications.

Three new sesquineolignans from the fruits of Crataegus pinnatifida

Chemical investigations of the 75% EtOH extract from the fruits of Crataegus pinnatifida led to the isolation of three undescribed naturally occurring sesquineolignans with two 8-O-4'-type neolignan moieties, cratapinnatifidas A-C (1–3). Their chemical structures were elucidated by the comprehensive spectroscopic analyses (HRESIMS, 1D and 2D-NMR). In the bioactivity assay, their cytotoxic activities against two human hepatoma cell lines (HepG2 and Hep3B) were evaluated and no significant activity was seen with IC50 values ranging from 12.5 to 50.0 μM.

Mutation of EPO 5'UTR Facilitates Interaction with HIF2 and Causes Autosomal Dominant Erythrocytosis

We studied 10 affected and 11 non affected relatives of a five generation kindred with autosomal dominant familial erythrocytosis. We have excluded other known inherited forms of erythrocytosis. i.e., mutations of globin, the 2,3 DPG generating PBGM gene causing increased Hb/O2 affinity (low p50), gain-of-function mutations of erythropoietin receptor (EPOR), germ-line JAK2 mutations, and hypoxia inducible factor 2A (HIF2-A(EPAS1)),PHD2(EGLN1), and VHL mutations associated with augmented oxygen-sensing pathway. Those affected family members had moderately increased erythropoietin (EPO) levels, no splenomegaly, normal leukocyte and platelet numbers and normal p50 (presented at this mtg, Blood. 2003;102,162b). We sequenced whole exomes and adjacent portions of introns of two affected individuals and found a novel heterozygous 5`UTR EPO variant with change -136 nt upstream from the ATG EPO initiation site (NG_021471 -136 G&amp;gt;A). This variant segregated with the erythrocytosis phenotype in 15 relatives examined: the 7 affected subjects were heterozygous for this variant and the 8 unaffected were negative, suggesting its causative role in erythrocytosis (presented at this mtg, Blood. 2013;122,950). Other authors (NEJM 2018; 378:924) reported a variant of autosomal dominant familial erythrocytosis with a different EPO mutation: a single-nucleotide deletion (c.32delG) in exon 2 of the EPO gene causing a frameshift and alternative EPO mRNA transcripts, leading to increased production of functional EPO protein with shortened signal peptide and a novel N-terminus as cause of their familial erythrocytosis. In order to characterize function of our 5`UTR EPO variant, we introduced it into the EPO producing human hepatoma cell line Hep3B using CRISPR/Cas9 editing system by homologous recombination with single-stranded donor oligonucleotides. The targeted cells were sorted in 96 well plates (20 cells per well) and then each well tested for presence of -136 G&amp;gt;A variant by allele-specific PCR. We identified 3 heterozygous Hep3B for EPO-136 G&amp;gt;A; the second round of targeting generated homozygous Hep3B clones. The EPO mRNA of homozygous recombinants was greatly increased and detected even in normoxia, unlike non-edited Hep3B cells. No alternative EPO mRNA transcripts were detected in the engineered and non-edited Hep3B cells. To emulate human phenotype, the supernatants of cultured three heterozygousEPO-136 G&amp;gt;A Hep3B single-cell derived clones and controls in normoxic and hypoxic conditions were used to detect production of EPO. The hypoxic treatment increased ~2x the production of EPO from edited clones compared to non-edited Hep3B cells. The secreted EPO from heterozygousEPO-136 G&amp;gt;A Hep3B clones supported growth of EPO-dependent BaF3-EPOR cells more than supernatants from non-edited Hep3B cells. We then measured EPO transcript levels in Hep3B with EPO-136 G&amp;gt;A and parental Hep3B cell lines in normoxia and hypoxia. The hypoxia increased the relative expression of EPO-136 G&amp;gt;A allele in all three targeted heterozygousEPO-136 G&amp;gt;A Hep3B cell lines. The EPO gene promoter was largely unmethylated in both wild and mutated clones. To evaluate activity of this mutant EPO promoter, we sub-cloned wild and mutated 5`UTR EPO sequence upstream of luciferase reporter gene and transfected them into two EPO producing cell lines - Hep3B, Hep2G. The mutant significantly increased activity of the reporter. To study the interaction of EPO-136 G&amp;gt;A mutant promoter with HIF2 (principal transcription factor regulating EPO), we co-transfected EPO-luc reporter with HIF2-A expression plasmid. The activity of reporter with mutated EPO-136 G&amp;gt;A was further increased in these cells with augmented HIF2 levels. Indeed, alignment tools predicted the EPO-136 G&amp;gt;A genomic region as putative HIF2 binding site. This suggests that mutated 5`UTR of EPO augments interaction with HIF2, leading to increase production of EPO. Chromatin immunoprecipitation experiments are ongoing to model the transcriptional regulatory network accounting for augmented transcriptional regulation of this 5`UTR EPO gene variant. Here we report a novel mechanism of inherited erythrocytosis caused by increased transcription of mutated 5`UTR of EPO. Supported by Czech HRC, grant NV19-07-00412 and Ministry of Education, grant LTAUSA17142 and University of Utah. Disclosures No relevant conflicts of interest to declare.

Integrated Transcriptomic and Proteomic Analyses Reveal Role of the Hexosamine Biosynthetic Pathway in Invasion and Metastasis of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a major global cause of mortality. The epithelial-mesenchymal transition (EMT) transcription factor TWIST1 has been implicated in the invasion and metastasis of HCC, but the mechanism is unclear. Integrated transcriptomic and proteomic analyses on primary liver tumors and lung metastases from a spontaneous Twist1-dependent metastasis mouse model of MYC-induced HCC identified the hexosamine biosynthetic pathway (HBP) as a cancer cell autonomous mechanism for EMT-mediated invasion and metastasis, which was further validated in vitro and ex vivo. RNA-sequencing and mass spectrometry-based proteomics were carried out on primary liver tumors, lung metastases, normal livers and normal lungs from a Twist1-dependent metastasis mouse model of MYC-induced HCC (LMT); a metastatic MYC-induced HCC mouse model dependent on Twist1 phosphorylation mimetic mutant (LMT-DQD); a non-metastatic mouse model of MYC-induced HCC (LM); a non-metastatic MYC-induced HCC mouse model with the Twist box mutant (LMT-F191G); and wildtype mice. Data was analyzed in R. Organoids were derived from LM liver tumors, embedded into collagen and imaged using time-lapse DIC microscopy. Cell lines were derived from LM and LMT liver tumors, and along with human HCC cell lines Huh7, Hep3B and HepG2, were utilized for in vitro migration and invasion assays. 91 genes were differentially expressed between lung metastases versus primary liver tumors; primary liver tumors versus normal liver; and normal lung versus normal liver with p<0.05. Gene Ontology and KEGG Pathway Enrichment analyses revealed an enrichment of metabolic processes. Gfpt2, the gene encoding for the rate-limiting enzyme of the hexosamine biosynthetic pathway, was overexpressed >2 fold in metastases compared to primary tumor (p = 0.007). Kaplan-Meier estimates from cBioPortal show that HCC patients with genetic alterations in GFPT2 and/or its isoform GFPT1 have significantly worse overall median survival compared to patients without these alterations (27.5 months vs 83.24 months, p = 0.006). Functional validation studies demonstrated that increased O-GlcNAcylation by pharmacologic treatment with TMG or genetic manipulation by GFPT2 overexpression was sufficient to increase migration and invasion in vitro in a panel of human and murine HCC cell lines. Decreased HBP-O-GlcNAcylation by pharmacologic treatment with DON or genetic manipulation by GFPT2 shRNA knockdown inhibited migration and invasion in vitro. Similarly, using a novel organoid invasion assay, TMG treatment increased invasion of organoids derived from LM tumors ex vivo 2-fold (p<0.03) while DON treatment inhibited invasion 2-fold (p<0.03). The hexosamine biosynthetic pathway is required and sufficient for invasion and metastasis in vitro in HCC cell lines and ex vivo in HCC tumor-derived organoids. The HBP may be a potential therapeutic target for advanced HCC patients.