Hepa1-6 Cells Research Articles

Metal-organic framework IRMOFs coated with a temperature-sensitive gel delivering norcantharidin to treat liver cancer.

BACKGROUNDNorcantharidin (NCTD) is suitable for the treatment of primary liver cancer, especially early and middle primary liver cancer. This compound can reduce tumors and improve immune function. However, the side effects of NCTD have limited its application. There is a marked need to reduce the side effects and increase the efficacy of NCTD.AIMTo develop a nanomaterial carrier, NCTD-loaded metal-organic framework IRMOF-3 coated with a temperature-sensitive gel (NCTD-IRMOF-3-Gel), aiming to improve the anticancer activity of NCTD and reduce the drug dose.METHODSNCTD-IRMOF-3-Gel was obtained by a coordination reaction. The apparent characteristics and in vitro release of NCTD-IRMOF-3-Gel were investigated. Cell cytotoxicity assays, flow cytometry, and apoptosis experiments in mouse hepatoma (Hepa1-6) cells were used to determine the anti-liver cancer activity of NCTD-IRMOF-3-Gel in in vitro models.RESULTSThe particle size of NCTD-IRMOF-3-Gel was 50-100 nm, and the particle size distribution was uniform. The release curve showed that NCTD-IRMOF-3-Gel had an obvious sustained-release effect. The cytotoxicity assays showed that the free drug NCTD and NCTD-IRMOF-3-Gel treatments markedly inhibited Hepa1-6 cell proliferation, and the inhibition rate increased with increasing drug concentration. By flow cytometry, NCTD-IRMOF-3-Gel was observed to block the Hepa1-6 cell cycle in the S and G2/M phases, and the thermosensitive gel nanoparticles may inhibit cell proliferation by inducing cell cycle arrest. Apoptosis experiments showed that NCTD-IRMOF-3-Gel induced the apoptosis of Hepa1-6 cells.CONCLUSIONOur results indicated that the NCTD-IRMOF-3-Gel may be beneficial for liver cancer disease treatment.

Abstract 2894: Intracellular gap formation of the liver sinusoidal endothelial cells facilitates the liver metastasis ability of cancer cells

Abstract Engraftment of cancer cells to liver pre-metastatic niches and their subsequent growth requires complex interaction with liver sinusoidal endothelial cells (LSECs). Intracellular gap formation of LSECs results from the destruction of the fenestration under histopathological conditions, but their role in liver metastasis is not fully understood. Therefore, this study aims to clarify the molecular function of the gap formation in liver metastasis. To study gap formation, acetaminophen (APAP) and thioacetamide (TAA) were used to pretreat C57BL/6 wild-type mice by intraperitoneal injection. Then, 2 x 106 mouse hepatoma Hepa1-6 cells and colon cancer Colon-26 cells were intrasplenically injected in both treated and non-treated groups. Mice were sacrificed after 18 hours and 72 hours of injection to observe the cancer cell invasion in the hepatic sinusoidal lumen and to count the number of cancer cell engraftment in the liver, respectively. Hepa1-6 cells and isolated primary mouse LSECs were co-cultured for 18 hours to observe the phenomenon in vitro. Quantitative-PCR, Hematoxylin & Eosin, immunofluorescence staining, scanning electron microscope (SEM), transmission electron microscope (TEM), and 3 dimensions tomographic reconstruction image (3D-TRI) were conducted to analyze the data. After 72 hours of injection, the number of cancer cells engrafted into the liver was significantly increased in the APAP-injected group compared to the control (p<0.05). In the TAA model, defenestration of LSECs appeared in the fibrotic area whereas gap formation appeared in the non-fibrotic area, cancer cells dominantly engrafted into the non-fibrotic area. In vitro, LSECs co-cultured with Hepa1-6 increased mRNA levels of matrix metalloproteinase-9 (30-fold) and intercellular adhesion molecule 1 (1.5-fold) and depolymerization of F-actin. Simultaneously, the number of fenestrae and gap formation in LSECs was significantly increased compared to monoculture. Both in vivo-, in vitro- SEM and TEM images clearly showed that Hepa1-6 cells induced the gap formation in LSECs and their protrusions extended to the gaps. Furthermore, 3D-TRI reconstructed the image of cancer cells that broke through the LSEC wall and intracellularly invaded the hepatocyte. Our study demonstrated that the formation of intracellular gaps in LSECs promotes the invasion of cancer cells into the liver. This new insight may lead to novel strategies to prevent liver metastasis. Citation Format: Truong Huu Hoang, Misako Sato-Matsubara, Yuasa Hideto, Tsutomu Matsubara, Hayato Urushima, Thuy Thi Thanh Le, Atsuko Daikoku, Yoshinori Okina, Katsutoshi Yoshizato, Norifumi Kawada, Akihiro Tamori. Intracellular gap formation of the liver sinusoidal endothelial cells facilitates the liver metastasis ability of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2894.

Abstract 1437: E7386, a CREB binding protein (CBP)/β-catenin interaction inhibitor, suppresses the hypoxic response induced by angiogenesis inhibition in hepatocellular carcinoma models

Abstract Background: Aberrant activation of Wnt/β-catenin signaling plays an important role in both carcinogenesis and modulation of the tumor microenvironment. E7386, an investigational anti-cancer agent, inhibits the binding of β-catenin to its transcriptional co-activator, CREB binding protein (CBP), thereby modulating Wnt/β-catenin signaling. We previously reported that E7386 suppressed tumor growth in preclinical mouse models with aberrant activation of Wnt/β-catenin signaling. Moreover, the combination of E7386 plus lenvatinib, a multiple receptor tyrosine kinase inhibitor that mainly targets vascular endothelial growth factor receptors and fibroblast growth factor receptors, showed stronger antitumor and antiangiogenic activities than each treatment alone. Here, we investigated the molecular mechanism of the combination activities of E7386 plus lenvatinib. Methods: We examined antitumor activity of E7386 plus lenvatinib in multiple human tumor xenograft and mouse syngeneic models. Tumor samples underwent RNA sequencing (RNA-seq) followed by weighted gene correlation network analysis. Furthermore quantitative PCR was used to assess changes in the expression of some hypoxia-related genes. The protein interaction between β-catenin and HIF1 was analyzed by immunoprecipitation-western blotting. Results: RNA-seq analysis of the effects of E7386, lenvatinib, and E7386 plus lenvatinib on the tumor microenvironment in the Hepa1-6 hepatocellular carcinoma syngeneic tumor model in mice showed that lenvatinib suppressed the expression of angiogenesis-related genes and that E7386 plus lenvatinib enhanced this suppression. Although lenvatinib increased the expression of hypoxia-related genes, E7386 plus lenvatinib attenuated this phenomenon. According to these results, we hypothesized that E7386 inhibits the formation of the β-catenin/HIF-1 complex. To confirm the effect of E7386 on the hypoxic response, we conducted an in vitro quantitative PCR assay under hypoxic conditions in Hepa1-6 cells and the human hepatocellular carcinoma cell lines, HepG2 and Hep3B. In accordance with the RNA-seq result, E7386 significantly suppressed the expression levels of hypoxia-related genes in the HepG2 and Hep3B cells. Finally, data from immunoprecipitation-western blotting demonstrated that β-catenin formed a complex with HIF-1 and that E7386 clearly inhibited this complex formation in HepG2 cells. Conclusion: These results suggest that E7386 enhances angiogenesis inhibition when used in combination with lenvatinib and that E7386 suppresses the hypoxic response induced by angiogenesis inhibition. Further analysis is required to clarify the precise mechanisms of actions of the combination of E7386 plus lenvatinib. Citation Format: Mizuki Kimura, Yusaku Hori, Megumi Kuronishi, Takayuki Kimura, Ryoga Ishida, Kenji Ichikawa, Yoichi Ozawa, Yu Kato. E7386, a CREB binding protein (CBP)/β-catenin interaction inhibitor, suppresses the hypoxic response induced by angiogenesis inhibition in hepatocellular carcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1437.

Abstract 1739: Intratumoral INTASYL™ self-delivering RNAi targeting PD-1 provides in vivo tumor control and mechanistic modulation of tumor microenvironment analogous to that of systemic anti-PD-1 antibody

Abstract The clinical success of immune checkpoint blockade (ICB) antibodies like those inhibiting PD-1 have revolutionized cancer care, however, durable responses are still limited. Systemic ICB can also elicit immune-related adverse effects (irAEs) whereas local intratumoral (IT) delivery has the potential to mitigate these toxicities. The INTASYL™ platform is a self-delivering RNAi technology that (1) provides efficient delivery into target cells without specialized formulations, mechanical perturbation, or drug delivery systems; and (2) specifically and durably silences target gene expression and (3) has been shown to be safe and effective in clinical applications following local administration. Here we demonstrate that IT treatment with the PD-1 targeting INTASYL PH-762 inhibits tumor growth in a dose dependent fashion in two tumor models, Hepa1-6 and CT26. Modulation of key immune cell populations in the tumor microenvironment (TME) by systemic antibody PD-1 inhibition are recapitulated under IT treatment with the PD-1 targeting INTASYL PH-762. Hepa1-6 or CT26 tumor cells were implanted subcutaneously into female C57BL/6 or BALB/c mice, respectively. Vehicle, non-targeting control (NTC-647) or murine PD-1 targeting INTASYL (mPH-762) were each administered IT at doses ranging from 0.02 - 2 mg/dose; murine anti-PD-1 monoclonal antibody (anti-PD-1 mAb) was administered intraperitoneally (IP) at 200 μg/dose, each on Days 1, 3, 7, 10 and 14. Tumor volumes and body weight were recorded. Tumors (N = 6/group) were isolated from the Hepa1-6 model for ex vivo analyses. All treatments were well tolerated. Treatment with mPH-762 inhibited tumor growth in Hepa1-6 in a dose dependent manner compared to PBS or NTC, with doses of 0.5 mg or 2 mg inhibiting tumor growth similar to anti-PD-1 mAb. In the TME, treatment with mPH-762 modulated tumor immune populations toward antitumor phenotypes, including significantly increasing overall %CD45+ cells and CD8+ T cells, reducing putative Tregs, and increasing median M1/M2 tumor associated macrophage ratios compared to PBS or NTC-647 in a dose associated manner and similar to anti-PD-1 mAb. Dose-correlating on-target silencing of PD-1 protein expression was observed under treatment with mPH-762 but not anti-PD-1 mAb across key TME cell populations. In conclusion, in vivo tumor control elicited by the PD-1 silencing INTASYL mPH-762 (IT) was akin to that provided by systemic anti-PD-1 mAb (IP). Similar modulation of TME immune cell populations associated with antitumor efficacy were observed under both systemic Ab and local INTASYL (IT) treatments. As INTASYL is efficacious and may mitigate irAEs caused by antibody ICB, INTASYL PH-762 warrants further investigation in patients. ​ Citation Format: Benjamin Cuiffo, Melissa Maxwell, Dingxue Yan, James Cardia, Simon P. Fricker. Intratumoral INTASYL™ self-delivering RNAi targeting PD-1 provides in vivo tumor control and mechanistic modulation of tumor microenvironment analogous to that of systemic anti-PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1739.

Enhancing effect of Panax ginseng on Zip4-mediated zinc influx into the cytosol

BackgroundZinc homeostasis is essential for human health and is regulated by several zinc transporters including ZIP and ZnT. ZIP4 is expressed in the small intestine and is important for zinc absorption from the diet. We investigated in the present study the effects of Panax ginseng (P. ginseng) extract on modulating Zip4 expression and cellular zinc levels in mouse Hepa cells. MethodsHepa cells were transfected with a luciferase reporter plasmid that contains metal-responsive elements, incubated with P. ginseng extract, and luciferase activity was measured. Using 65ZnCl2, zinc uptake in P. ginseng-treated cells was measured. The expression of Zip4 mRNA and protein in Hepa cells was also investigated. Finally, using a luciferase reporter assay system, the effects of several ginsenosides were monitored. ResultsThe luciferase activity in cells incubated with P. ginseng extract was significantly higher than that of control cells cultured in normal medium. Hepa cells treated with P. ginseng extract exhibited higher zinc uptake. P. ginseng extract induced Zip4 mRNA expression, which resulted in an enhancement of Zip4 protein expression. Furthermore, some ginsenosides, such as ginsenoside Rc and Re, enhanced luciferase activity driven by intracellular zinc levels. ConclusionP. ginseng extract induced Zip4 expression at the mRNA and protein level and resulted in higher zinc uptake in Hepa cells. Some ginsenosides facilitated zinc influx. On the basis of these results, we suggest a novel effect of P. ginseng on Zip4-mediated zinc influx, which may provide a new strategy for preventing zinc deficiency.

The Combination Immunotherapy of TLR9 Agonist and OX40 Agonist via Intratumoural Injection for Hepatocellular Carcinoma.

BackgroundThe response rate of immunotherapy via immune checkpoint blockade in hepatocellular carcinoma (HCC) is limited due to multiple immune evasion mechanisms. OX40 is a T cell co-stimulating molecule which suppresses the cancer immune evasion by activating effector T cells (Teffs) and counteracting regulatory T cells (Tregs). TLR9 belongs to the toll-like receptor superfamily which promotes tumour antigen presentation by stimulating the maturation of dendritic cells. Though the combination immunotherapy of TLR9 agonist (CpG) and OX40 agonist (anti-OX40 antibody) has shown encouraging efficacy in various tumours, its effect on HCC remains unknown.Materials and MethodsOrthotopic and ectopic HCC models were constructed by implanting Hepa1-6 cells at different body sites of the mice. Immune agents were administrated via three ways, including intratumoural injection into one site of the tumour, intraperitoneal injection, and subcutaneous injection. The anti-tumour immune response was evaluated by the regression of both the local treated tumour and distant untreated tumour. The ratio and function of CD4+ T cells, CD8+ T cells, Tregs and myeloid-derived suppressor cells (MDSCs) were analyzed by flow cytometry.ResultsCpG via intratumoural injection remarkably upregulated the weakly expressed OX40 of intratumoural T cells. The combination immunotherapy of CpG and anti-OX40 antibody via intratumoural injection significantly inhibited the growth of local and distant tumours, and also effectively prevented their recurrence. Excitingly, drug administration via intratumoural injection, rather than via intraperitoneal or subcutaneous injections, induced potent anti-tumour immune response. Furthermore, we demonstrated that the combination immunotherapy promoted CD8+ and CD4+ T cells, and inhibited Tregs and myeloid-derived suppressor cells, contributing to the effective inhibition on HCC. Noteworthily, the combination immunotherapy also induced an immune memory response.ConclusionThe intratumoural administration of combined CpG and anti-OX40 antibody serves as a promising immunotherapy against HCC.

3,3'-Diindolylmethane Exhibits Significant Metabolism after Oral Dosing in Humans.

3,3'-Diindolylmethane (DIM), a major phytochemical derived from ingestion of cruciferous vegetables, is also a dietary supplement. In preclinical models, DIM is an effective cancer chemopreventive agent and has been studied in a number of clinical trials. Previous pharmacokinetic studies in preclinical and clinical models have not reported DIM metabolites in plasma or urine after oral dosing, and the pharmacological actions of DIM on target tissues is assumed to be solely via the parent compound. Seven subjects (6 males and 1 female) ranging from 26-65 years of age, on a cruciferous vegetable-restricted diet prior to and during the study, took 2 BioResponse DIM 150-mg capsules (45.3 mg DIM/capsule) every evening for one week with a final dose the morning of the first blood draw. A complete time course was performed with plasma and urine collected over 48 hours and analyzed by UPLC-MS/MS. In addition to parent DIM, two monohydroxylated metabolites and 1 dihydroxylated metabolite, along with their sulfate and glucuronide conjugates, were present in both plasma and urine. Results reported here are indicative of significant phase 1 and phase 2 metabolism and differ from previous pharmacokinetic studies in rodents and humans, which reported only parent DIM present after oral administration. 3-((1H-indole-3-yl)methyl)indolin-2-one, identified as one of the monohydroxylated products, exhibited greater potency and efficacy as an aryl hydrocarbon receptor agonist when tested in a xenobiotic response element-luciferase reporter assay using Hepa1 cells. In addition to competitive phytochemical-drug adverse reactions, additional metabolites may exhibit pharmacological activity highlighting the importance of further characterization of DIM metabolism in humans. SIGNIFICANCE STATEMENT: 3,3'-Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, is an effective cancer chemopreventive agent in preclinical models and a popular dietary supplement currently in clinical trials. Pharmacokinetic studies to date have found little or no metabolites of DIM in plasma or urine. In marked contrast, we demonstrate rapid appearance of mono- and dihydroxylated metabolites in human plasma and urine as well as their sulfate and glucuronide conjugates. The 3-((1H-indole-3-yl)methyl)indolin-2-one metabolite exhibited significant aryl hydrocarbon receptor agonist activity, emphasizing the need for further characterization of the pharmacological properties of DIM metabolites.

NRF2 DLG Domain Mutations Identified in Japanese Liver Cancer Patients Affect the Transcriptional Activity in HCC Cell Lines.

Geographically, East Asia had the highest liver cancer burden in 2017. Besides this, liver cancer-related deaths were high in Japan, accounting for 3.90% of total deaths. The development of liver cancer is influenced by several factors, and genetic alteration is one of the critical factors among them. Therefore, the detailed mechanism driving the oncogenic transformation of liver cells needs to be elucidated. Recently, many researchers have focused on investigating the liver cancer genome and identified somatic mutations (MTs) of several transcription factors. In this line, next-generation sequencing of the cancer genome identified that oxidative stress-related transcription factor NRF2 (NFE2L2) is mutated in different cancers, including hepatocellular carcinoma (HCC). Here, we demonstrated that NRF2 DLG motif mutations (NRF2 D29A and L30F), found in Japanese liver cancer patients, upregulate the transcriptional activity of NRF2 in HCC cell lines. Moreover, the transcriptional activity of NRF2 mutations is not suppressed by KEAP1, presumably because NRF2 MTs disturb proper NRF2-KEAP1 binding and block KEAP1-mediated degradation of NRF2. Additionally, we showed that both MTs upregulate the transcriptional activity of NRF2 on the MMP9 promoter in Hepa1-6 and Huh7 cells, suggesting that MT derived gain-of-function of NRF2 may be important for liver tumor progression. We also found that ectopic overexpression of oncogenic BRAF WT and V600E increases the transcriptional activity of NRF2 WT on both the 3xARE reporter and MMP9 promoter. Interestingly, NRF2 D29A and L30F MTs with oncogenic BRAF V600E MT synergistically upregulate the transcription activity of NRF2 on the 3xARE reporter and MMP9 promoter in Hepa1-6 and Huh7 cells. In summary, our findings suggest that MTs in NRF2 have pathogenic effects, and that NRF2 MTs together with oncogenic BRAF V600E MT synergistically cause more aberrant transcriptional activity. The high activity of NRF2 MTs in HCC with BRAF MT warrants further exploration of the potential diagnostic, prognostic, and therapeutic utility of this pathway in HCC.

Farnesoid X Receptor Mediates Tumor Immune Evasion by Targeting PD-L1 Induced by a Novel Bile Acid in Hepatocellular Carcinoma

Background: Emerging evidence indicates that bile acids (BAs) play pivotal roles in hepatocellular carcinoma (HCC). Farnesoid X receptor (FXR), the receptor of BAs, is considered to be a regulator of the immune response and inflammation in various diseases. However, the relevance of the immune microenvironment to FXR in HCC is unknown. Methods: Flow cytometry analyses were performed to measure the apoptosis rates of HCC cells and the immune checkpoint changes of immune cells. Kaplan-Meier analysis with log-rank test for assessing overall survival and time to recurrence was performed. Exosomes were isolated and cocultured with CD4+ T and CD8+ T cells for functional assays. HCC models were established in a gene-deficient mouse strain to evaluate FXR capability. Findings: We identified a previously unrecognized protumorigenic bile acid subset, Norcholic acid (NorCA). Mechanistic studies suggested that NorCA promotes tumorigenesis and the development of HCC cells by regulating the FXR-SHP-PD-L1 axis. Moreover, tumor-derived Exos formed by NorCA induction affected the immune microenvironment by regulating CD4+ T cells. In addition, we identified a subgroup of FXRlowPD-L1high patients, who have a poor prognosis. Furthermore, a drug combination study demonstrated that the FXR agonist (GW4064) exerted synergistic effects with an anti‐PD‐1 antibody (Ab) in a mouse model. Interpretation: Our results identify a novel protumorigenic bile acid subset demonstrates the immune function of FXR in HCC cells, offering valuable evidence for using FXR agonists combined with an anti‐PD‐1 Ab in combating HCC. Funding: Research supported by grants from fifteen funds. All sources of funding were declared at the end of the text. Declaration of Interest: None potential conflicts of interest were disclosed. Ethical Approval: This study was approved by the Ethics Review Board of the Third Affiliated Hospital, Sun Yat-sen University. Huh-7, LM3, and Hepa1-6 cells were obtained from ATCC

Suppression of E4BP4 SUMOylation sensitize mice to HFD‐induced fatty liver disease by promoting lipid droplet formation

Background As the most common chronic liver disease, non-alcoholic fatty liver disease (NAFLD) affects 10-30% of the general population. Characterized by excessive lipid droplet formation in hepatocytes, fatty liver may progress to nonalcoholic steatohepatitis (NASH), eventually leading to cirrhosis and liver failure. Currently, there have been no effective therapeutic or preventive measures for NAFLD. Thus, there is an urgent need to identify hepatic mediators that promote lipid droplet accumulation as well as the underlying molecular mechanisms in the liver during the pathogenesis of NAFLD. We previously reported that E4 promoter-binding protein 4 (E4BP4) is an insulin-induced stabilizer of the lipogenic factor SREBP-1c and promotes the SREBP-1c-mediated lipogenesis in hepatocytes. Our most recent mass spectrometry data revealed that E4BP4 may undergo SUMOylation, a reversible post-translational modification that was shown to regulate E4BP4-dependent NK cell development. In this study, we set out to determine how hepatic E4BP4 regulates lipid droplet formation and liver steatosis a high-fat diet-induced NAFLD mouse model and investigate how E4BP4 SUMOylation impacts the process. Results Compared with E4bp4flox/flox mice, E4bp4 liver-specific KO (E4bp4-LKO) mice exhibit decreased lipid accumulation in the liver despite similar body weight after 12 weeks of high-fat diet feeding. Our microarray analysis showed a reduction in the expression of lipid droplet biding genes such as Cidea, Cidec (Fsp27), Plin4 in the liver of E4bp4-LKO mice. Restoring E4bp4 expression in E4bp4-LKO primary hepatocytes was sufficient to induce lipid droplet formation and Fsp27β expression, while overexpression of Fsp27β increased lipid droplets and triglycerides in E4bp4-LKO primary mouse hepatocytes and promoted lipid accumulation HFD-fed E4bp4-LKO mice. Meanwhile, we generated an E4BP4 mutant, namely E4BP4-5KR, which contains five lysine to arginine (5KR) substitutions in the five SUMOylation motifs. Our in vitro SUMOylation assay confirmed the SUMOylation of E4BP4-WT but not E4BP4-5KR mutant in the presence of E1/2, SUMO2/3, and ATP. We detected more lipid droplet formation in hepa1 cells transfected with E4bp4-5KR vs. E4bp4-WT. Lastly, the abundance of SUMOylated E4BP4 was reduced in the liver of high-fat diet-fed mice vs. regular chow-fed mice. Conclusion All together, our data demonstrated that E4bp4 drives lipid droplet formation and live steatosis in high-fat diet-fed mice likely through its regulation of lipid-droplet binding genes. Our study also highlights the critical role of deSUMOylation of hepatic E4BP4 in promoting NAFLD and suggests that E4BP4 SUMOylation in the liver could be targeted for treating NAFLD.

Paternal obesity impairs hepatic gluconeogenesis of offspring by altering Igf2/H19 DNA methylation

Over the past four decades, the global prevalence of obesity has increased rapidly in all age ranges. Emerging evidence suggests that paternal lifestyle and environmental exposure have a crucial role in the health of offspring. Therefore, the current study investigated the impact of paternal obesity on the metabolic profile of offspring in a male mouse model of obesity. Female offspring of obese fathers fed a high-fat diet (HFD) (60% kcal fat) showed hyperglycemia because of enhanced gluconeogenesis and elevated expression of phosphoenolpyruvate carboxykinase (PEPCK), which is a key enzyme involved in the regulation of gluconeogenesis. Methylation of the Igf2/H19 imprinting control region (ICR) was dysregulated in the liver of offspring, and the sperm, of HFD fathers, suggesting that epigenetic changes in germ cells contribute to this father-offspring transmission. In addition, we explored whether H19 might regulate hepatic gluconeogenesis. Our results showed that overexpression of H19 in Hepa1-6 cells enhanced the expression of PEPCK and gluconeogenesis by promoting nuclear retention of forkhead box O1 (FOXO1), which is involved in the transcriptional regulation of Pepck. Thus, the current study suggests that paternal exposure to HFD impairs the gluconeogenesis of offspring via altered Igf2/H19 DNA methylation.

Effective and safe delivery of GLP-1AR and FGF-21 plasmids using amino-functionalized dual-mesoporous silica nanoparticles in vitro and in vivo

Nanomaterials have attracted increased attention because of their excellent drug-carrying capacity. However, these nanomaterials are rarely used in the treatment of metabolic diseases. Liraglutide, a glucagon-like peptide-1 receptor agonist, has been widely used in the treatment of type 2 diabetes mellitus (T2DM). Furthermore, fibroblast growth factor 21 (FGF-21) has been found to improve glucose metabolism and insulin resistance (IR). To investigate whether these two molecules have synergistic effects in vivo, we developed a novel drug delivery system using amino-functionalized and embedded dual-mesoporous silica nanoparticles (N-EDMSNs) to simultaneously carry liraglutide and FGF-21, and observed their biological effects. The resultant N-EDMSNs possessed unique hierarchical porous structures consisting of open large pores (>10 nm) and small mesopores (~2.5 nm) in the silica framework, highly positively charged surfaces and good disperisity in aqueous solution. We found that N-EDMSNs had a high loading capacity for exogenous genes and low toxicity to Hepa1-6 cells. Moreover, N-EDMSNs can simultaneously carry FGF-21 plasmids and liraglutide and successfully transfect them into Hepa1-6 cells. The transfection efficiency of N-EDMSNs was higher than that of Lipofectamine 2000 in vitro. In mice experiments, N-EDMSNs/pFGF21 treatment resulted in higher FGF-21 expression in the liver than pFGF21 treatment with hydrodynamic delivery. Compared with both pFGF21 and liraglutide, N-EDMSNs/pFGF21/Lira treatment significantly reduced the food intake, body weight, and blood glucose; increased the energy expenditure and improved hepatic IR in high-fat diet (HFD)-fed mice. Our results demonstrated that the biological effects of N-EDMSNs/pFGF21/Lira complexes were better than those of pFGF21 combined with liraglutide in vivo.

Manganese Ferrite Nanoparticles Enhance the Sensitivity of Hepa1-6 Hepatocellular Carcinoma to Radiation by Remodeling Tumor Microenvironments.

We evaluated the effect of manganese ferrite nanoparticles (MFN) on radiosensitization and immunologic responses using the murine hepatoma cell line Hepa1-6 and the syngeneic mouse model. The clonogenic survival of Hepa1-6 cells was increased by hypoxia, while being restricted by ionizing radiation (IR) and/or MFN. Although MFN suppressed HIF-1α under hypoxia, the combination of IR and MFN enhanced apoptosis and DNA damage in Hepa1-6 cells. In the Hepa1-6 syngeneic mouse model, the combination of IR and MFN notably limited the tumor growth compared to the single treatment with IR or MFN, and also triggered more frequent apoptosis in tumor tissues than that observed under other conditions. Increased expression of PD-L1 after IR was not observed with MFN alone or the combination of IR and MFN in vitro and in vivo, and the percentage of tumor-infiltrating T cells and cytotoxic T cells increased with MFN, regardless of IR, in the Hepa1-6 syngeneic mouse model, while IR alone led to T cell depletion. MFN might have the potential to overcome radioresistance by alleviating hypoxia and strengthening antitumor immunity in the tumor microenvironment.

25380 Cholecystokinin-B Receptor -Mediates Growth of Hepatocellular Carcinoma

ABSTRACT IMPACT: Cholecystokinin-B Receptor -Mediates Growth of Hepatocellular Carcinoma with the use proglumide. Proglumide is a non-selective antagonistic drug therefore, strategies that block signaling at the CCK-BR may provide to be a novel therapeutic option for Hepatocellular Carcinoma treatment OBJECTIVES/GOALS: Cholecystokinin (CCK)and gastrin mediate the growth of Hepatocellular Carcinoma (HCC) through CCK-R and interruption of this signaling pathway could decrease HCC. CCK-Receptors are overexpressed in HCC and proliferation may be mediated through CCK-B. Blockade of the CCK-BR with proglumide decreased both growth in vitro and tumor growth in vivo. METHODS/STUDY POPULATION: RNA was extracted from murine Hepa1-6, RIL-175 and human HepG2 cells and was evaluated by qRT-PCR for expression of CCK-AR, CCK-BR and gastrin. CCK-R protein expression was analyzed by flow cytometry. HCC cells were treated in vitro with CCK peptide, the CCK-AR antagonist or the CCK-BR antagonist. Proliferation of selective CCK-R KO cells was compared to that of wild-type cells. To determine the effect of a CCK-R antagonist on tumor growth in vivo two cohorts of mice bearing subcutaneous Hepa1-6 or RIL-175 HCC tumors were treated with an oral bioavailable CCK-R antagonist proglumide or untreated water for 3-4 weeks. The mice bearing Hepa1-6 tumors were placed on a high-fat diet to raise blood CCK levels. Mice bearing RIL-175 tumors were fed standard chow to determine if proglumide could block autocrine growth by gastrin. RESULTS/ANTICIPATED RESULTS: The mRNA expression of CCK-AR, CCK-BR and gastrin were increased 80-90-fold in all HCC cell lines compared to that of normal liver. CCK-BRs were detected on >85% of the cells by flow cytometry. CCK peptide (1nM) stimulated HCC growth in vitro in both wild-type cells and in CCK-AR KO cells but not in CCK-BR KO cells. CCK-BR antagonist blocked CCK-stimulated growth in vitro but the CCK-AR antagonist did not, suggesting that the CCK-BR was responsible for mediating proliferation. In vivo tumor growth was significantly reduced with proglumide treatment by 70% (p<0.05) in Hepa1-6 and by 73% (p<0.001) in RIL-75 tumors, respectively. DISCUSSION/SIGNIFICANCE OF FINDINGS: CCK-Rs are overexpressed in HCC and proliferation appears to be mediated through the CCK-BR. Downregulation with CRISPR Cas9 or blockade of the CCK-BR with an antagonist decreases growth in vitro and proglumide therapy decreases tumor growth in vivo. Strategies that block signaling at the CCK-BR maybe a novel therapeutic option for HCC treatment.

Development of a genetically modified hepatoma cell line with heat-inducible high liver function.

The online version of this article (10.1007/s10616-021-00457-4) contains supplementary material, which is available to authorized users.

Immunotherapy of Tumor RNA-Loaded Lipid Nanoparticles Against Hepatocellular Carcinoma.

PurposeHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Most current therapeutic strategies primarily include localized treatment, lacking effective systemic strategies. Meanwhile, recent studies have suggested that RNA vaccines can effectively activate antigen-presenting cells (APCs) and lymphocytes to produce a strong systemic immune response and inhibit tumor growth. However, tumor vaccines loaded with a single tumor antigen may induce immunosuppression and immune evasion, while identifying tumor-specific antigens can require expensive and laborious procedures. Therefore, the use of whole tumor cell antigens are currently considered to be promising, potentially effective, methods. Previously, we developed a targeted liposome-polycation-DNA (LPD) complex nanoparticle that possess a small size, high RNA encapsulation efficiency, and superior serum stability. These particles were found to successfully deliver RNA to tumor sites. In the current study, we encapsulated total tumor-derived RNA in lipid nanoparticles (LNPs) to target dendritic cells (DCs) to incite expeditious and robust anti-tumor immunity.MethodsTotal tumor-derived RNA was extracted from liver cancer cells (Hepa1-6 cells). LNPs loaded with tumor RNA were then prepared thin-film hydration method. The ability of RNA LNPs to induce DC maturation, cytotoxicity, and anti-tumor activity, was investigated in vitro and in vivo.ResultsThe average particle size of LNPs and RNA LNPs was 102.22 ± 4.05 nm and 209.68 ± 6.14 nm, respectively, while the zeta potential was 29.97 ± 0.61 mV and 42.03 ± 0.42 mV, respectively. Both LNPs and RNA LNP vaccines exhibited good distribution and stability. In vitro, RNA LNP vaccines were capable of promoting DC maturation and inducing T lymphocytes to kill Hepa1-6 cells. In vivo, RNA LNP vaccines effectively prevent and inhibit HCC growth.ConclusionRNA LNPs may serve as an effective antigen specific vaccine to induce anti-tumor immunity for HCC.

Nuclear factor Y participates in alcoholic liver disease by activating SREBP1 expression in mice

Chronic and excessive alcohol consumption leads to alcoholic liver disease (ALD). However, the molecular mechanisms in the regulation of ALD have not been fully deciphered. Liver lipid accumulation is an important research direction in ALD. In this study, the physiological role of nuclear factor Y (NF–Y) in ALD and the related mechanisms were investigated using murine hepatocytes and an ethanol-induced liver injury mouse model. In this study, ethanol promoted hepatic NF–Y expression in a mouse model and Hepa1-6 mouse hepatocytes. Lentivirus-mediated NF–Y overexpression in Hepa1-6 cells markedly increased sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FASN) expression compared with empty vector control cells. Conversely, CRISPR/Cas9-mediated knockdown of NF–Y subunit A (NF-YA) attenuated FASN and SREBP1 expression. Mechanistically, luciferase reporter gene assays and chromatin immunoprecipitation (ChIP) analysis indicated that NF–Y activates the transcription of SREBP1 by directly binding to the CCAAT regulatory sequence motif in the promoter. Overall, our results reveal a previously unrecognized physiological function of NF–Y in ALD by activating sterol regulatory element-binding protein 1 (SREBP1). Modulation of hepatic NF–Y expression may therefore offer an attractive therapeutic approach to manage ALD.

ApoM is an important potential protective factor in the pathogenesis of primary liver cancer.

In recent years, abnormal liver lipid metabolism has emerged as one of the important pathogenesis pathways of primary liver cancer. It is highly important to identify the mechanisms to explore potential prevention and treatment targets. Apolipoprotein M is specifically expressed in the liver and participates in liver lipid metabolism, but the evidence that ApoM affects primary liver cancer is insufficient. The Cancer Genome Atlas (TCGA) database and clinical case analysis, as well as animal level and cell level analysis suggest that the expression level of ApoM gene in cancer tissues is lower than that in paracarcinoma tissues. Further experimental research found that the deletion of ApoM significantly increased the proliferation of mouse liver cancer cells (Hepa1-6) and inhibited the level of apoptosis induced by cisplatin. In addition, mouse liver cancer cells lacking ApoM showed stronger migration and invasion capabilities in transwell experiments. In contrast, overexpression of ApoM in Hepa1-6 cells and Huh-7 cells showed an inhibition of proliferation, up-regulation apoptosis and reduced migration and invasion. In vivo, the deletion of the ApoM accelerated tumorigenesis in nude mice and allowed the mice to develop liver tumor mutations more quickly under the induction of N-nitrosodiethylamine and the survival time of mice was shorter than that control. Therefore, ApoM may be a potential protective factor to inhibit the occurrence and development of primary liver cancer.

A Pegylated Flavin Adenine Dinucleotide PEG Complex to Boost Immunogenic and Therapeutic Effects in a Liver Cancer Model.

Flavin adenine dinucleotide (FAD) is engaged in several metabolic diseases. Its main role is being a cofactor essential for the activity of many flavoproteins, which play a crucial role in electron transport pathways in living systems. The aim of this study was to apply a pegylated flavins formulation named FAD-PEG diacide complex as theranostic pathway in cancer therapy. For this purpose, a mouse liver cancer model induced by Hepa1-6 cells was used to evaluate the therapeutic efficacy of FAD (named NP1) and FAD-PEG diacide complex (named NP2). The cytokines were applied to screen the serum inflammatory factors, to establish the blood cell content of different groups of nude mice. The highlights follows that FAD formulations (NP1; NP2) significantly suppressed the tumor growth and reduced the tumor index without effects on the body weight of mice. Furthermore, NP2 significantly reduced the serum levels of cytokines IL-6, TNF-α and IL-12 (P70). The reported results provide the proof-of-concept for the synthesis of a smart adjuvant for liver cancer therapy and support their further development in the field of nanomedicine.

6-Gingerol Ameliorates Hepatic Steatosis via HNF4α/miR-467b-3p/GPAT1 Cascade.

Background & AimsThe development of nonalcoholic fatty liver disease (NAFLD) can be modulated by microRNAs (miRNA). Dietary polyphenols modulate the expression of miRNA such as miR-467b-3p in the liver. In addition, 6-gingerol (6-G), the functional polyphenol of ginger, has been reported to ameliorate hepatic steatosis; however, the exact mechanism involved and the role of miRNA remain elusive. In this study, we assessed the role of miR-467b-3p in the pathogenesis of hepatic steatosis and the regulation of miR-467b-3p by 6-G through the hepatocyte nuclear factor 4α (HNF4α).MethodsmiR-467b-3p expression was measured in free fatty acid (FFA)-treated hepatocytes or liver from high-fat diet (HFD)-fed mice. Gain- or loss-of-function of miR-467b-3p was induced using miR-467b-3p–specific miRNA mimic or miRNA inhibitor, respectively. 6-G was exposed to FFA-treated cells and HFD-fed mice. The HNF4α/miR-467b-3p/GPAT1 axis was measured in mouse and human fatty liver tissues.ResultsWe found that miR-467b-3p was down-regulated in liver tissues from HFD-fed mice and in FFA-treated Hepa1-6 cells. Overexpression of miR-467b-3p decreased intracellular lipid accumulation in FFA-treated hepatocytes and mitigated hepatic steatosis in HFD-fed mice via negative regulation of glycerol-3-phosphate acyltransferase-1 (GPAT1). In addition, miR-467b-3p up-regulation by 6-G was observed. 6-G inhibited FFA-induced lipid accumulation and mitigated hepatic steatosis. Moreover, it increased the transcriptional activity of HNF4α, resulting in the increase of miR-467b-3p and subsequent decrease of GPAT1. HNF4α/miR-467b-3p/GPAT1 signaling also was observed in human samples with hepatic steatosis.ConclusionsOur findings establish a novel mechanism by which 6-G improves NAFLD. This suggests that targeting of the HNF4α/miR-467b-3p/GPAT1 cascade may be used as a potential therapeutic strategy to control NAFLD.