AdipoR1 Regulates Ionizing Radiation-Induced Ferroptosis in HCC Cells Through Nrf2/xCT Pathway

Background: Ferroptosis is a type of cell death accompanied by iron-dependent lipid peroxidation, however, how IR-induced ferroptosis is regulated in Hepatocellular carcinoma cells (HCC) remains largely unknown. We have previously found that adiponectin receptor 1(AdipoR1) might be a prognostic biomarker for HCC after stereotactic body radiotherapy (SBRT). In this study, we aimed to elucidate the roles of AdipoR1 in radiation-induced Ferroptosis of HCC.Methods: Human HCC cell line MHCC-97H and HepG2 and human hepatic cell lines LO2 were tested. qRT-PCR and western blotting were used to detect mRNA and protein expression respectively, colony formation assay was used to evaluate the radiosensitivity and flow cytometry was used to assess lipid peroxidation and cell death. Dual-Luciferase Reporter assay system was used to detect the transcription activity. Results: Ionizing Radiation (IR) upregulated the expression of AdipoR1 in HCC cells and AdipoR1 knockdown could promote radiation sensitivity of HCC cells. AdipoR1 knockdown could decrease the expression of Nrf2 and Nrf2 protein stability. Nrf2 could bind to xCT promoter and promoted the transcription and expression of xCT. AdipoR1 knockdown increased significantly lipid peroxidation and ferroptosis induced by IR or Erastin respectively, which could be abolished by overexpression of Nrf2 and xCT.Conclusion: AdipoR1 knockdown can promote radiation sensitivity of HCC cells; AdipoR1 regulates IR-induced cell death by AdipoR1-Nrf2-xCT pathway.

Cytotoxicity and Mitochondrial Effects of Phenolic and Quinone-Based Mitochondria-Targeted and Untargeted Antioxidants on Human Neuronal and Hepatic Cell Lines: A Comparative Analysis

Mitochondriotropic antioxidants (MC3, MC6.2, MC4 and MC7.2) based on dietary antioxidants and analogs (caffeic, hydrocaffeic, trihydroxyphenylpropanoic and trihydroxycinnamic acids) were developed. In this study, we evaluate and compare the cytotoxicity profile of novel mitochondria-targeted molecules (generally known as MitoCINs) on human HepG2 and differentiated SH-SY5Y cells with the quinone-based mitochondria-targeted antioxidants MitoQ and SkQ1 and with two non-targeted antioxidants, resveratrol and coenzyme Q10 (CoQ10). We further evaluate their effects on mitochondrial membrane potential, cellular oxygen consumption and extracellular acidification rates. Overall, MitoCINs derivatives reduced cell viability at concentrations about six times higher than those observed with MitoQ and SkQ1. A toxicity ranking for both cell lines was produced: MC4 < MC7.2 < MC3 < MC6.2. These results suggest that C-6 carbon linker and the presence of a pyrogallol group result in lower cytotoxicity. MC3 and MC6.2 affected the mitochondrial function more significantly relative to MitoQ, SkQ1, resveratrol and CoQ10, while MC4 and MC7.2 displayed around 100–1000× less cytotoxicity than SkQ1 and MitoQ. Based on the mitochondrial and cytotoxicity cellular data, MC4 and MC7.2 are proposed as leads that can be optimized to develop safe drug candidates with therapeutic application in mitochondrial oxidative stress-related diseases.

Cyclin-dependent kinase 19 upregulation correlates with an unfavorable prognosis in hepatocellular carcinoma

Objectives Cyclin-dependent kinase 19 (CDK19) is a component of the mediator coactivator complex, which is required for transcriptional activation. In this study, we utilized public databases and wet-bench hepatic cell line experiments to elucidate the potential roles of CDK19 in hepatocellular cancer (HCC). Materials and methods We studied the relationships between CDK19 expression and several clinical features related to HCC via the Oncomine and UALCAN databases. The prognostic value of CDK19 was tested using the Kaplan–Meier Plotter database. We presented the mutations of CDK19 and addressed the relation of CDK19 expression with immune cell infiltration by means of the cBioPortal, Catalogue of Somatic Mutations in Cancer (COSMIC) and Tumor IMmune Estimation Resource (TIMER) databases. Hub genes were obtained and further analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. To test the in silico findings, we knocked down CDK19 with short hairpin RNA (shRNA) technology in two hepatic cell lines and conducted several functional characterization experiments. Results Marked CDK19 upregulation was found in HCC tissues versus normal liver tissues, and CDK19 mRNA expression had high diagnostic value in HCC patients. Subgroup analysis showed that CDK19 overexpression was associated with sex, tumor stage and TP53 mutation status. The prognostic value of CDK19 upregulation for overall survival (OS) was significant in patients with stage 2–3, stage 3–4, and grade 2 disease. One percent of the patients had CDK19 mutations, but no relationship between CDK19 mutation and prognosis was observed. CDK19 was positively correlated with the abundances of CD4 + T cells, macrophages and dendritic cells. We identified 10 genes correlated with CDK19, 8 of which presented excellent prognostic value in HCC. These hub genes were directly involved in cell division and regulation of the G2/M cell cycle transition. Protein–protein interaction (PPI) and pathway predictions indicated that CDK19 is highly likely to be involved in several cellular functions, such as proliferation, migration, and invasion. These functions were strongly interfered from two independent hepatic cell lines after CDK19 knockdown. Conclusions CDK19 could be a prognostic marker in HCC, and its therapeutic potential in HCC needs further study.

Cyclin Dependent Kinase 19 Upregulation Correlates With Unfavorable Prognosis in Hepatocellular Carcinoma

Objectives: Cyclin dependent kinase 19 (CDK19) is a component of the Mediator co-activator complex, which is required for transcriptional activation. In this study, we will utilize the public data and combine it with wet-bench experiments in hepatic cell lines to elucidate the potential roles of CDK19 in hepatocellular cancer (HCC).Materials and Methods: We studied the relationships between CDK19 expression and several clinical features related with HCC by consulting Oncomine and UALCAN. The prognostic value of CDK19 was tested using the Kaplan‐Meier Plotter database. We presented the mutations of CDK19 and addressed its relations with immune cells with the use of cBioPortal, and COSMIC and TIMER database. Hub genes were obtained and further analysed using the STRING database. To test the in silico findings, we knocked down CDK19 with short hairpin RNA (shRNA) technology in two hepatic cell lines, and then several functional characterization experiments were conducted. Results: A remarkably higher level of CDK19 expression was found in HCC tissues than normal liver tissues, and CDK19 mRNA expression has high diagnostic value in HCC patients. Subgroup analysis showed that CDK19 overexpression were associated with gender, tumor stage and TP53 mutant. Prognostic values of CDK19 upregulation for overall survival (OS) were significant in patients with stage 2-3, stage 3-4, grade 2 and etc. 1% of the patients have mutations at CDK19, and we did not observe a potential relationship between CDK19 mutation and prognosis. CDK19 showed positive correlations with the abundances of CD4+ T cells, macrophages and dendritic cells. We identified 10 genes that correlated with CDK19, 8 of which presented excellent prognostic value in HCC. Besides, these hub genes were directly involved in cell division and regulation of G2/M transition of mitotic cell cycle. PPI and pathway predictions indicated that CDK19 should have a high possibility to be involved with several cellular functions, such as proliferation, migration, and invasion. These functions were strongly interfered in two independent hepatic cell lines, after knocking down CDK19. Conclusions: CDK19 could serve as a prognostic marker in HCC and it deserves further work to test its therapeutic potential to HCC.

Human-Induced Pluripotent Stem Cell-Derived Hepatocytes and their Culturing Methods to Maintain Liver Functions for Pharmacokinetics and Safety Evaluation of Pharmaceuticals

Human hepatocytes are essential cell types for pharmacokinetics and the safety evaluation of pharmaceuticals. However, widely used primary hepatocytes with individual variations in liver function lose those functions rapidly in culture. Hepatic cell lines are convenient to use but have low liver functions. Human-Induced Pluripotent Stem (hiPS) cells can be expanded and potentially differentiated into any cell or tissue, including the liver. HiPS cell-derived Hepatocyte-Like Cells (hiPSHeps) are expected to be extensively used as consistent functional human hepatocytes. Many laboratories are investigating methods of using hiPS cells to differentiate hepatocytes, but the derived cells still have immature liver functions. In this paper, we describe the current uses and limitations of conventional hepatic cells, evaluating the suitability of hiPS-Heps to pharmacokinetics and the safety evaluation of pharmaceuticals, and discuss the potential future use of non-conventional non-monolayer culture methods to derive fully functional hiPS-Heps.

Iron Oxide Nanoparticle-Induced Autophagic Flux Is Regulated by Interplay between p53-mTOR Axis and Bcl-2 Signaling in Hepatic Cells

Iron oxide-based nanoparticles have been repeatedly shown to affect lysosomal-mediated signaling. Recently, nanoparticles have demonstrated an ability to modulate autophagic flux via lysosome-dependent signaling. However, the precise underlying mechanisms of such modulation as well as the impact of cellular genetic background remain enigmatic. In this study, we investigated how lysosomal-mediated signaling is affected by iron oxide nanoparticle uptake in three distinct hepatic cell lines. We found that nanoparticle-induced lysosomal dysfunction alters sub-cellular localization of pmTOR and p53 proteins. Our data indicate that alterations in the sub-cellular localization of p53 protein induced by nanoparticle greatly affect the autophagic flux. We found that cells with high levels of Bcl-2 are insensitive to autophagy initiated by nanoparticles. Altogether, our data identify lysosomes as a central hub that control nanoparticle-mediated responses in hepatic cells. Our results provide an important fundamental background for the future development of targeted nanoparticle-based therapies.

SAT-726 Estrogen Receptor Alpha as a Potential Target for Bisphenol A-Mediated Epigenetic Reprogramming: An in Vitro Analysis

Perinatal exposure to bisphenol A (BPA) has been shown to reprogram the hepatic epigenome of rodents and may promote the development of various metabolic diseases later in life, such as nonalcoholic fatty liver disease (NAFLD). This developmental reprogramming is characterized by the creation of “super promoters” at target genes implicated in metabolic pathways. While it is unclear how these “super promoters” are created, their creation is potentially mediated through BPA and estrogen receptor (ER) interaction. In order to test this potential mechanism, in vitro methods were used to examine ER target gene expression via RT-qPCR in 2 human hepatic cell lines transiently transfected with the ER isoform, ER alpha, prior to BPA exposure for various lengths of time. Within individual time points, there were no significant differences in target gene expression levels between cells that had been transfected with ER alpha and the vector control. Gene expression levels in the target genes were visibly increased at the 24-hour exposure mark in both transfection groups in comparison to the 0- and 6-hour time points, however only a fraction of these increases were found to be statistically significant. These gene expression patterns are not only consistent with previous studies examining target gene expression in BPA-treated hepatic cell lines, but more importantly, suggest BPA does not act via ER alpha to orchestrate the epigenetic changes seen in vitro. BPA may interact with a different ER isoform or an unknown target to create the observed “super promoters” at target genes, reinforcing the promiscuity of BPA and other xenoestrogens in facilitating epigenetic modifications, and ultimately, disease phenotypes.

Estrogen stimulates SREBP2 expression in hepatic cell lines via an estrogen response element in the SREBP2 promoter

Objective Hypoestrogenism in women is strongly associated with menopause and it can lead to lipid disorder, which predisposes people to premature cardiovascular disease. However, the mechanism of lipid disorder remains unclear. Sterol regulatory element-binding protein 2 (SREBP2) is the key transcription factor regulating cholesterol metabolism. We hypothesize that estrogen regulates SREBP2 transcription through an estrogen response element (ERE) in the SREBP2 promoter region. Methods Human hepatoblastoma cells (HepG2) were treated with dose-dependent concentrations of estradiol (E2) for 24 h. Then, SREBP2 expression was determined via real-time PCR and immunofluorescence. The expressions of the SREBP2 downstream target genes HMGCR and LDLR were determined via real-time PCR. Lipid secretion in the culture media of HepG2 cells was measured using ELISA. Through bioinformatics analysis, we identified high-scoring ERE-like sequences in the SREBP2 gene promoter. Chromatin immunoprecipitation analysis was used to confirm the ERE. DNA fragments of the putative or mutated ERE-like sequence were synthesized and ligated into pGL3-basic plasmid to construct the SREBP2 promoter luciferase reporter systems. SREBP2-Luciferase (SREBP2-Luc), SREBP2-Mutation (SREBP2-Mut) and the blank control were transfected into hepatic cell lines. Luciferase activities were measured using the dual-luciferase reporter assay system. Chromatin immunoprecipitation analysis and the luciferase reporter assay were repeated in human hepatoma cells (HuH-7). Results We found that E2 dose-dependently increased the expression of SREBP2 in HepG2 cells and that the increased levels were blocked when treated with an estrogen receptor-alpha antagonist. Additionally, E2 increased both HMGCR and LDLR expression and lipid secretion in HepG2 cells. Notably, we identified a functional ERE in the SREBP2 gene promoter, to which E2 could specifically bind and induce transcription. Conclusions An ERE was identified in the SREBP2 gene promoter. It mediates the regulation of SREBP2 expression by estrogen in hepatocytes. This study provides a mechanism to link cardiovascular disease with estrogen.

Comparison between Lipofectamine RNAiMAX and GenMute transfection agents in two cellular models of human hepatoma

RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-transfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have higher viability with respect to Lipofectamine RNAiMAX-administered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.