Transcriptomics based identification of S100A3 as the key anti-hepatitis B virus factor of 16F16.

More than 250 million people worldwide have chronic hepatitis B virus (HBV) infections, resulting in over 1 million annual fatalities because HBV cannot be adequately treated with current antivirals. Hepatocellular carcinoma (HCC) risk is elevated in the presence of the HBV. Novel and powerful medications that specifically target the persistent viral components are needed to remove infection. This study aimed to use HepG2.2.15 cells and the rAAV-HBV1.3 C57BL/6 mouse model established in our laboratory to examine the effects of 16F16 on HBV. The transcriptome analysis of the samples was performed to examine the impact of 16F16 therapy on host factors. We found that the HBsAg and HBeAg levels significantly decreased in a dose-dependent manner following the 16F16 treatment. 16F16 also showed significant anti-hepatitis B effects in vivo. The transcriptome analysis showed that 16F16 regulated the expression of several proteins in HBV-producing HepG2.2.15 cells. As one of the differentially expressed genes, the role of S100A3 in the anti-hepatitis B process of 16F16 was further investigated. The expression of the S100A3 protein significantly decreased following the 16F16 therapy. And upregulation of S100A3 caused an upregulation of HBV DNA, HBsAg, and HBeAg in HepG2.2.15 cells. Similarly, knockdown of S100A3 significantly reduced the levels of HBsAg, HBeAg, and HBV DNA. Our findings proved that S100A3 might be a new target for combating HBV pathogenesis. 16F16 can target several proteins involved in HBV pathogenesis, and may be a promising drug precursor molecule for the treatment of HBV.

Inhibitory Effect of Styrylpyrone Extract of Phellinus linteus on Hepatic Steatosis in HepG2 Cells.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is estimated to be approximately about 25.24% of the population worldwide. NAFLD is a complex syndrome and is characterized by a simple benign hepatocyte steatosis to more severe steatohepatitis in the liver pathology. Phellinus linteus (PL) is traditionally used as a hepatoprotective supplement. Styrylpyrone-enriched extract (SPEE) obtained from the PL mycelia has been shown to have potential inhibition effects on high-fat- and high-fructose-diet-induced NAFLD. In the continuous study, we aimed to explore the inhibitory effects of SPEE on free fatty acid mixture O/P [oleic acid (OA): palmitic acid (PA); 2:1, molar ratio]-induced lipid accumulation in HepG2 cells. Results showed that SPEE presented the highest free radical scavenging ability on DPPH and ABTS, and reducing power on ferric ions, better than that of partitions obtained from n-hexane, n-butanol and distilled water. In free-fatty-acid-induced lipid accumulation in HepG2 cells, SPEE showed an inhibition effect on O/P-induced lipid accumulation of 27% at a dosage of 500 µg/mL. As compared to the O/P induction group, the antioxidant activities of superoxide dismutase, glutathione peroxidase and catalase were enhanced by 73%, 67% and 35%, respectively, in the SPEE group. In addition, the inflammatory factors (TNF-α, IL-6 and IL-1ß) were significantly down-regulated by the SPEE treatment. The expressions of anti-adipogenic genes involved in hepatic lipid metabolism of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) were enhanced in the SPEE supplemented HepG2 cells. In the protein expression study, p-AMPK, SIRT1 and PGC1-α were significantly increased to 121, 72 and 62%, respectively, after the treatment of SPEE. Conclusively, the styrylpyrone-enriched extract SPEE can ameliorate lipid accumulation and decrease inflammation and oxidative stress through the activation of SIRT1/AMPK/PGC1-α pathways.

Mallotucin D, a Clerodane Diterpenoid from Croton crassifolius, Suppresses HepG2 Cell Growth via Inducing Autophagic Cell Death and Pyroptosis.

Hepatocellular carcinoma (HCC) is a major subtype of primary liver cancer with a high mortality rate. Pyroptosis and autophagy are crucial processes in the pathophysiology of HCC. Searching for efficient drugs targeting pyroptosis and autophagy with lower toxicity is useful for HCC treatment. Mallotucin D (MLD), a clerodane diterpenoid from Croton crassifolius, has not been previously reported for its anticancer effects in HCC. This study aims to evaluate the inhibitory effects of MLD in HCC and explore the underlying mechanism. We found that the cell proliferation, DNA synthesis, and colony formation of HepG2 cells and the angiogenesis of HUVECs were all greatly inhibited by MLD. MLD caused mitochondrial damage and decreased the TOM20 expression and mitochondrial membrane potential, inducing ROS overproduction. Moreover, MLD promoted the cytochrome C from mitochondria into cytoplasm, leading to cleavage of caspase-9 and caspase-3 inducing GSDMD-related pyroptosis. In addition, we revealed that MLD activated mitophagy by inhibiting the PI3K/AKT/mTOR pathway. Using the ROS-scavenging reagent NAC, the activation effects of MLD on pyroptosis- and autophagy-related pathways were all inhibited. In the HepG2 xenograft model, MLD effectively inhibited tumor growth without detectable toxicities in normal tissue. In conclusion, MLD could be developed as a candidate drug for HCC treatment by inducing mitophagy and pyroptosis via promoting mitochondrial-related ROS production.

Close association between the synergistic toxicity of zearalenone-deoxynivalenol combination and microRNA221-mediated PTEN/PI3K/AKT signaling in HepG2 cells.

Mycotoxins can impart different types of combined toxicity to humans and animals, therefore, it is critical to understand the underlying mechanisms to eliminate the harm. Herein a combination of zearalenone (ZEA) at 2 µM and deoxynivalenol (DON) at 0.1 µM decreased cell viability and increased ROS level in HepG2 cells, suggesting synergistic toxicity exerted by ZEA and DON even at their low toxic concentrations. Moreover, apoptosis and inflammatory response were promoted after the co-exposure of ZEA and DON, indicated by the increased expression of BAX, Caspase-3, IL-1ß and IL-6 genes. Such synergistic toxicity was closely associated with miR-221-mediated PTEN/PI3K/AKT signal pathway, with a negative regulatory relationship between PTEN and PI3K/AKT signaling. MiR-221 could influence cell viability and ROS level to counter the combined toxicity of ZEA and DON through targeting directly PTEN gene. This study demonstrated the toxicological impact of mycotoxin interactions on cells, and critical role of the interplay between miRNAs and PTEN in monitoring the synergistic toxicity of mycotoxin mixture.

Hyperuricemia induces lipid disturbances by upregulating the CXCL-13 pathway.

The molecular mechanism underlying hyperuricemia-induced lipid metabolism disorders is not clear. The purpose of the current study was to investigate the mechanism of lipid disturbances in a hyperuricemia mice model. RNA-Seq showed that differentially expressed genes (DEGs) in the fatty acid synthesis signaling pathway were mainly enriched and CXCL-13 was significantly enriched in protein-protein interaction networks. Western blotting, Q-PCR, and immunofluorescence results further showed that hyperuricemia upregulated CXCL-13 and disturbed lipid metabolism in vivo and in vitro. Furthermore, CXCL-13 alone also promoted the accumulation of lipid droplets and upregulated the expression of FAS and SREBP1, blocking AMPK signaling and activating the PKC and P38 signaling pathways. Silencing CXCL-13 reversed uric-acid-induced lipid droplet accumulation, which further downregulated FAS and SREBP1 expression, inhibited the p38 and PKC signaling, and activated AMPK signaling. In conclusion, hyperuricemia induces lipid metabolism disorders via the CXCL-13 pathway, making CXCL-13 a key regulatory factor linking hyperuricemia and lipid metabolism disorders. These results may provide novel insights for the treatment of hyperuricemia.NEW & NOTEWORTHY The underlying molecular mechanism of hyperuricemia-induced lipid metabolism disorders is still unclear. The study aimed to investigate the mechanism of lipid disturbance in hyperuricemia mice model. To our knowledge, we proposed for the first time that CXCL-13 may be a key regulator of hyperuricemia and lipid metabolism disorders. These results may provide new insights for the clinical treatment of hyperuricemia.

Bombyxin II Regulates Glucose Absorption and Glycogen Synthesis through the PI3K Signaling Pathway in HepG2 Cells.

Bombyxin, as an insulin-like insect hormone, was discovered in the silkmoth Bombyx mori. It can regulate the metabolism of trehalose and glycogen in Bombyx mori, but whether it has glucose absorption and glycogen synthesis effect on mammalian cells was not clear. BombyxinII (BbxII) and mutant BbxII (mBbxII) genes were cloned into pcDNA3.1(+) vector, respectively; then, gene vectors were transfected into 293FT cells using Lipofectamine 2000. Levels of mRNA and protein expression of BbxII and mBbxII were detected by PCR and Western blot in 293FT cells, respectively. Glucose consumption and glycogenesis were determined by glucose oxidase-peroxidase (GOD-POD) and periodic acid-Schiff (PAS) staining in HepG2 cells; the PI3K signaling pathway was inhibited with wortmannin S1952 in HepG2 cells. Result showed that BbxII and mBbxII genes were being successfully expressed in 293FT cells, respectively. The expression protein of BbxII gene is 10kd pre-bombyxinII, and yet, the expression protein of mBbxII gene is 4kd mature bombyxinII. Only the 4kd bombyxinII showed increased glucose uptake and glycogenesis in HepG2 cells, and the ability of increasing glucose uptake was equal to the human insulin (10 nM). PI3K-wortmannin S1952 inhibitor can decrease the glycogen synthesis induced by bombyxin II protein in HepG2 cells. In conclusion, mature bombyxin II may adjust glucose absorption and glycogen synthesis in HepG2 cells through the PI3K signaling pathway.

Identifying Circular RNAs in HepG2 Expressing Genotype IV Swine Hepatitis E Virus ORF3 Via Whole Genome Sequencing.

Swine hepatitis E (SHE) is a new type of zoonotic infectious disease caused by swine hepatitis E virus (SHEV). Open reading frame 3 (ORF3) is a key regulatory and virulent protein of SHEV. Circular RNAs (circRNAs) are a special kind of non-coding RNA molecule, which has a closed ring structure. In this study, to identify the circRNA profile in host cells affected by SHEV ORF3, adenovirus ADV4-ORF3 mediated the overexpression of ORF3 in HepG2 cells, whole genome sequencing was used to investigate the differentially expressed circRNAs, GO and KEGG were performed to enrichment analyze of differentially expressed circRNA-hosting gene, and Targetscan and miRanda softwares were used to analyze the interaction between circRNA and miRNA. The results showed adenovirus successfully mediated the overexpression of ORF3 in HepG2 cells, 1,105 up-regulation circRNAs and 1,556 down-regulation circRNAs were identified in ADV4-ORF3 infection group compared with the control. GO function enrichment analysis of differentially expressed circRNAs-hosting genes classified three main categories (cellular component, biological process and molecular function). KEGG pathway enrichment analysis scatter plot showed the pathway term of top20. The circRNAs with top10 number of BS sites for qRT-PCR validation were selected to confirmed, the results indicated that the up-regulated hsa_circ_0001423 and hsa_circ_0006404, and down-regulated of hsa_circ_0004833 and hsa_circ_0007444 were consistent with the sequencing data. Our findings first preliminarily found that ORF3 protein may affect triglyceride activation (GO:0006642) and riboflavin metabolism (ko00740) in HepG2 cells, which provides a scientific basis for further elucidating the effect of ORF3 on host lipid metabolism and the mechanism of SHEV infection.

Arginine-Arginine-Leucine Peptide Targeting Heat Shock Protein 70 for Cancer Imaging.

Arg-Arg-Leu (RRL) is a potent tumor-homing tripeptide. However, the binding target is unclear. In this study, we intended to identify the binding target of RRL and evaluate the tumor targeting of 99mTc-MAG3-RRL in vivo. Biotin-RRL, 5-TAMRA-RRL, and 99mTc-MAG3-RRL were designed to trace the binding target and tumor lesion. Immunoprecipitation-mass spectrometry was conducted to identify the candidate proteins and determination of the subcellular localization was also performed. A pull-down assay was performed to demonstrate the immunoprecipitate. Fluorescence colocalization and cell uptake assays were performed to elucidate the correlation between the selected binding protein and RRL, and the internalization mechanism of RRL. Biodistribution and in vivo imaging were performed to evaluate the tumor accumulation and targeting of 99mTc-MAG3-RRL. The target for RRL was screened to be heat shock protein 70 (HSP70). The prominent uptake distribution of RRL was concentrated in the membrane and cytoplasm. A pull-down assay demonstrated the existence of HSP70 in the biotin-RRL captured complex. Regarding fluorescence colocalization and cell uptake assays, RRL may interact with HSP70 at the nucleotide-binding domain (NBD). Clathrin-dependent endocytosis and macropinocytosis could be a vital internalization mechanism of RRL. In vivo imaging and biodistribution both demonstrated that 99mTc-MAG3-RRL can trace tumors with satisfactory accumulation in hepatoma xenograft mice. The radioactive signals accumulated in tumor lesions can be blocked by VER-155008, which can bind to the NBD of HSP70. Our findings revealed that RRL may interact with HSP70 and that 99mTc-MAG3-RRL could be a prospective probe for visualizing overexpressed HSP70 tumor sections.

Cyanidin-3-O-glucoside and its metabolite protocatechuic acid ameliorate 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced cytotoxicity in HepG2 cells by regulating apoptotic and Nrf2/p62 pathways.

The present study investigated the protective effects and mechanism of action of cyanidin-3-O-glucoside (C3G) and its major metabolite protocatechuic acid (PCA) against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced cytotoxicity in HepG2 cells. The results demonstrated that C3G and PCA dose-dependently suppressed PhIP-induced mutation in Salmonella typhimurium TA98, and inhibited PhIP-induced cytotoxicity and apoptosis in HepG2 cells. Western blot analysis indicated that C3G and PCA minimized PhIP-induced cell damage by reversing the abnormal expression of Bax/Bcl-2, Cytochrome c, cleaved Caspase-3, XIAP, Nrf2, HO-1, LC3 and p62 involved in intrinsic apoptotic and Nrf2/p62 pathways. Molecular docking results revealed that C3G and PCA were able to interfere with Nrf2 signaling and apoptotic cascade through binding to Keap1 and Bcl-2. Moreover, the protective effect of C3G was stronger than that of PCA. These findings suggested that dietary consumption of food sources rich in C3G can fight against the health risks of heterocyclic aromatic amines.

Dual Effect of Taxifolin on ZEB2 Cancer Signaling in HepG2 Cells.

Polyphenols, secondary metabolites of plants, exhibit different anti-cancer and cytoprotective properties such as anti-radical, anti-angiogenic, anti-inflammation, or cardioprotective. Some of these activities could be linked to modulation of miRNAs expression. MiRNAs play an important role in posttranscriptional regulation of their target genes that could be important within cell signalling or preservation of cell homeostasis, e.g., cell survival/apoptosis. We evaluated the influence of a non-toxic concentration of taxifolin and quercetin on the expression of majority human miRNAs via Affymetrix GeneChip™ miRNA 3.0 Array. For the evaluation we used two cell models corresponding to liver tissue, Hep G2 and primary human hepatocytes. The array analysis identified four miRNAs, miR-153, miR-204, miR-211, and miR-377-3p, with reduced expression after taxifolin treatment. All of these miRNAs are linked to modulation of ZEB2 expression in various models. Indeed, ZEB2 protein displayed upregulation after taxifolin treatment in a dose dependent manner. However, the modulation did not lead to epithelial mesenchymal transition. Our data show that taxifolin inhibits Akt phosphorylation, thereby diminishing ZEB2 signalling that could trigger carcinogenesis. We conclude that biological activity of taxifolin may have ambiguous or even contradictory outcomes because of non-specific effect on the cell.

An antitumor peptide RS17-targeted CD47, design, synthesis, and antitumor activity.

BACKGROUND: CD47 is a widely expressed transmembrane protein located on the surface of somatic cells. It mediates a variety of cellular processes including apoptosis, proliferation, adhesion, and migration. An important role for CD47 is the transmission of a "Don't eat me" signal by interacting with SIRPα on the macrophage surface membrane, thereby preventing the phagocytosis of normal cells. However, cancer cells can take advantage of this autogenous signal to protect themselves from phagocytosis, thus enabling immune escape. Blocking the interaction between CD47 and SIRPα has proven to be effective in removing cancer cells. The treatment of various cancers with CD47 monoclonal antibodies has also been validated. METHODS: We designed and synthesized a peptide (RS17), which can specifically bind to CD47 and block CD47-SIRPα signaling. The affinity of RS17 for CD47-expressing tumor cells was determined, while the inhibition of CD47-SIRPα signaling was evaluated in vitro and in vivo. RESULTS: The results indicated that RS17 significantly promotes the phagocytosis of tumor cells by macrophages and had a similar therapeutic effect compared with a positive control (CD47 monoclonal antibodies). In addition, a cancer xenograft mouse model was established using CD47-expressing HepG2 cells to evaluate the effect of RS17 on tumor growth in vivo. Using ex vivo and in vivo mouse models, RS17 demonstrated a high inhibitory effect on tumor growth. CONCLUSIONS: Based on our results, RS17 may represent a novel therapeutic peptide for cancer therapy.

Synthesis, Anticancer Assessment, and Molecular Docking of Novel Chalcone-Thienopyrimidine Derivatives in HepG2 and MCF-7 Cell Lines.

Heterocycles containing thienopyrimidine moieties have attracted attention due to their interesting biological and pharmacological activities. In this research article, we reported the synthesis of a series of new hybrid molecules through merging the structural features of chalcones and pyridothienopyrimidinones. Our results indicated that the synthesis of chalcone-thienopyrimidine derivatives from the corresponding thienopyrimidine and chalcones proceeded in a relatively short reaction time with good yields and high purity. Most of these novel compounds exhibited moderate to robust cytotoxicity against HepG2 and MCF-7 cancer cells similar to that of 5-fluorouracil (5-FU). The results indicated that IC50 of the two compounds (3b and 3g) showed more potent anticancer activities against HepG2 and MCF-7 than 5-FU. An MTT assay and flow cytometry showed that only 3b and 3g had anticancer activity and antiproliferative activities at the G1 phase against MCF-7 cells, while six compounds (3a-e and 3g) had cytotoxicity and cell cycle arrest at different phases against HepG2 cells. Their cytotoxicity was achieved through downregulation of Bcl-2 and upregulation of Bax, caspase-3, and caspase-9. Although all tested compounds increased oxidative stress via increment of MDA levels and decrement of glutathione reductase (GR) activities compared to control, the 3a, 3b, and 3g in HepG2 and 3b and 3g in MCF-7 achieved the target results. Moreover, there was a positive correlation between cytotoxic efficacy of the compound and apoptosis in both HepG2 (R 2 = 0.531; P = 0.001) and MCF-7 (R 2 = 0.219; P = 0.349) cell lines. The results of molecular docking analysis of 3a-g into the binding groove of Bcl-2 revealed relatively moderate binding free energies compared to the selective Bcl-2 inhibitor, DRO. Like venetoclax, compounds 3a-g showed 2 violations from Lipinski's rule. However, the results of the ADME study also revealed higher drug-likeness scores for compounds 3a-g than for venetoclax. In conclusion, the tested newly synthesized chalcone-pyridothienopyrimidinone derivatives showed promising antiproliferative and apoptotic effects. Mechanistically, the compounds increased ROS production with concomitant cell cycle arrest and apoptosis. Therefore, regulation of the cell cycle and apoptosis are possible targets for anticancer therapy. The tested compounds could be potent anticancer agents to be tested in future clinical trials after extensive pharmacodynamic, pharmacokinetic, and toxicity profile investigations.

Apple polyphenol extract alleviates lipid accumulation in free-fatty-acid-exposed HepG2 cells via activating autophagy mediated by SIRT1/AMPK signaling.

Defective degradation of intracellular lipids induced by autophagy is causally linked to the development of non-alcoholic fatty liver disease (NAFLD). Natural agents that can restore autophagy could therefore have the potentials for clinical applications for this public health issue. Herein, we investigated the effects of apple polyphenol extract (APE) on fatty acid-induced lipids depositions in HepG2 cells. APE treatment alleviated palmitic acid and oleic acid-induced intracellular lipid accumulation, concomitant with the increased autophagy, restored lysosomal acidification, inhibited lipid synthesis and slight promotion of fatty acid oxidation. Mechanistically, APE up-regulated the expression of SIRT1, activated LKB1/AMPK pathway and inhibited mTOR signaling. Over-expressed or knock-down SIRT1 positively regulated AMPK and ATG7 expressions. SIRT1 and ATG7 knock-down impaired APE induction of improved lipid accumulation, increased intracellular TG content. Thus, APE induction of autophagy to ameliorate fatty acid-induced lipid deposition is SIRT1 dependent, APE conserved preventive potentials for clinical hepatosteatosis.

Liver X Receptor Alpha Activation Inhibits Autophagy and Lipophagy in Hepatocytes by Dysregulating Autophagy-Related 4B Cysteine Peptidase and Rab-8B, Reducing Mitochondrial Fuel Oxidation.

BACKGROUND AND AIMS: Fat accumulation results from increased fat absorption and/or defective fat metabolism. Currently, the lipid-sensing nuclear receptor that controls fat utilization in hepatocytes is elusive. Liver X receptor alpha (LXRα) promotes accumulation of lipids through the induction of several lipogenic genes. However, its effect on lipid degradation is open for study. Here, we investigated the inhibitory role of LXRα in autophagy/lipophagy in hepatocytes and the underlying basis. APPROACH AND RESULTS: In LXRα knockout mice fed a high-fat diet, or cell models, LXRα activation suppressed the function of mitochondria by inhibiting autophagy/lipophagy and induced hepatic steatosis. Gene sets associated with "autophagy" were enriched in hepatic transcriptome data. Autophagy flux was markedly augmented in the LXRα knockout mouse liver and primary hepatocytes. Mechanistically, LXRα suppressed autophagy-related 4B cysteine peptidase (ATG4B) and Rab-8B, responsible for autophagosome and -lysosome formation, by inducing let-7a and microRNA (miR)-34a. Chromatin immunoprecipitation assay enabled us to find LXRα as a transcription factor of let-7a and miR-34a. Moreover, 3' untranslated region luciferase assay substantiated the direct inhibitory effects of let-7a and miR-34a on ATG4B and Rab-8B. Consistently, either LXRα activation or the let-7a/miR-34a transfection lowered mitochondrial oxygen consumption rate and mitochondrial transmembrane potential and increased fat levels. In obese animals or nonalcoholic fatty liver disease (NAFLD) patients, let-7a and miR-34a levels were elevated with simultaneous decreases in ATG4B and Rab-8B levels. CONCLUSIONS: LXRα inhibits autophagy in hepatocytes through down-regulating ATG4B and Rab-8B by transcriptionally activating microRNA let-7a-2 and microRNA 34a genes and suppresses mitochondrial biogenesis and fuel consumption. This highlights a function of LXRα that culminates in the progression of liver steatosis and steatohepatitis, and the identified targets may be applied for a therapeutic strategy in the treatment of NAFLD.

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells.

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T overexpressing human NTCP, HNF4α, RXRα and PPARα) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines. HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.

Targeting protein palmitoylation decreases palmitate­induced sphere formation of human liver cancer cells.

Although non­alcoholic fatty liver disease (NAFLD) is considered a benign disorder, hepatic steatosis has been proposed to be involved in the tumorigenesis of liver cancer. However, the underlying mechanism for carcinogenesis in fatty liver diseases remains unclear. Cancer stem cells (CSCs) have been hypothesized to serve a key role in tumorigenesis. Tumor formation begins with a subset of heterogeneous cells that share properties with stem cells, such as self­renewal and undifferentiated properties. Our previous study reported that the saturated fatty acid palmitate (PA) significantly enhanced the CSC properties of the HepG2 human liver cancer cell line; however, its underlying mechanisms are unknown. In the present study, a proteomic approach was used to investigate the palmitoylation of proteins in HepG2 CSCs. CSC behavior was induced in HepG2 cells via 200 µM PA. Proteomic analysis was performed to identify post­transcriptional modifications of proteins in HepG2 CSCs in response to PA treatment. The present study identified proteins modified by palmitoylation in HepG2 CSC spheres formed following PA treatment. It was therefore hypothesized that palmitoylation may be crucial for CSC sphere formation. Furthermore, the present study demonstrated that two palmitoylation inhibitors, tunicamycin (5, 10 and 25 µg/ml) and 2­bromohexadecanoic acid (25, 50 and 150 µM), significantly decreased CSC sphere formation without affecting cell viability. An association was identified between sphere formation capacity and tumor­initiating capacity of CSCs. The results of the present study demonstrated that protein palmitoylation may influence the PA­induced CSC tumor­initiating capacity, and that the inhibition of palmitoylation may be a suitable chemopreventive strategy for treating patients with NAFLD.

Reactive oxygen species generation and mitochondrial dysfunction for the initiation of apoptotic cell death in human hepatocellular carcinoma HepG2 cells by a cyclic dipeptide Cyclo(-Pro-Tyr).

Cyclic dipeptides are increasingly gaining importance as considering its significant biological and pharmacological activities. This study was aimed to investigate the anticancer activity of a dipeptide Cyclo(-Pro-Tyr) (DP) identified from marine sponge Callyspongia fistularis symbiont Bacillus pumilus AMK1 and the underlying apoptotic mechanisms in the liver cancer HepG2 cell lines. MTT assay was done to demonstrate the cytotoxic effect of DP in HepG2 cells and mouse Fibroblast McCoy cells. Initially, apoptosis inducing activity of DP was identified using propidium iodide (PI) and acridine orange/ethidium bromide (AO/EB) dual staining, then it was confirmed by DNA fragmentation assay and western blotting analysis of apoptosis related markers Bax, Bcl-2, cytochrome c, caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Rhodamine 123 staining was performed to observe DP effects on the mitochondrial membrane potential (MMP) and DCFH-DA (Dichloro-dihydro-fluorescein diacetate) staining was done to measure the intracellular reactive oxygen species (ROS) levels. The MTT results revealed that DP initiated dose-dependent cytotoxicity in HepG2 cells, but no significant toxicity in mouse Fibroblast McCoy cells treated with DP at the specified concentrations. DP induced apoptosis, which is confirmed by the appearance of apoptotic bodies with PI and AO/EB dual staining, and DNA fragmentation. DP significantly elevated the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), enhanced cytochrome c release from mitochondria, increased caspase-3 activation, the cleavage of PARP and increased intracellular reactive oxygen species (ROS) levels. Besides this, DP successfully inhibited the phosphorylation of PI3K, AKT and increased PTEN expression. These results suggested DP might have anti-cancer effect by initiating apoptosis through mitochondrial dysfunction and downregulating PI3K/Akt signaling pathway in HepG2 cells with no toxicity effect on normal fibroblast cells. Therefore, DP may be developed as a potential alternative therapeutic agent for treating hepatocellular carcinoma.

Primary Human Hepatocytes, but Not HepG2 or Balb/c 3T3 Cells, Efficiently Metabolize Salinomycin and Are Resistant to Its Cytotoxicity.

Salinomycin is a polyether antibiotic showing anticancer activity. There are many reports of its toxicity to animals but little is known about the potential adverse effects in humans. The action of the drug may be connected to its metabolism. That is why we investigated the cytotoxicity of salinomycin and pathways of its biotransformation using human primary hepatocytes, human hepatoma cells (HepG2), and the mouse fibroblast cell line (Balb/c 3T3). The cytotoxicity of salinomycin was time-dependent, concentration-dependent, and cell-dependent with primary hepatocytes being the most resistant. Among the studied models, primary hepatocytes were the only ones to efficiently metabolize salinomycin but even they were saturated at higher concentrations. The main route of biotransformation was monooxygenation leading to the formation of monohydroxysalinomycin, dihydroxysalinomycin, and trihydroxysalinomycin. Tiamulin, which is a known inhibitor of CYP450 izoenzymes, synergistically induced cytotoxicity of salinomycin in all cell types, including non-metabolising fibroblasts. Therefore, the pharmacokinetic interaction cannot fully explain tiamulin impact on salinomycin toxicity.

Evaluation of the epigenetic alterations and gene expression levels of HepG2 cells exposed to zearalenone and α-zearalenol.

Zearalenone, produced by various Fusarium species, is a non-steroidal estrogenic mycotoxin that contaminates cereals, resulting in adverse effects on human health. We investigated the effects of zearalenone and its metabolite alpha zearalenol on epigenetic modifications and its relationship with metabolic pathways in human hepatocellular carcinoma cells following 24 h of exposure. Zearalenone and alpha zearalenol at the concentrations of 1, 10 and 50 µM significantly increased global levels of DNA methylation and global histone modifications (H3K27me3, H3K9me3, H3K9ac). Expression levels of the chromatin modifying enzymes EHMT2, ESCO1, HAT1, KAT2B, PRMT6 and SETD8 were upregulated by 50 µM of zearalenone exposure using PCR arrays, consistent with the results of global histone modifications. Zearalenone and alpha zearalenol also changed expression levels of the AhR, LXRα, PPARα, PPARÉ£, L-fabp, LDLR, Glut2, Akt1 and HK2 genes, which are related to nuclear receptors and metabolic pathways. PPARÉ£, a key regulator of lipid metabolism, was selected from among these genes for further analysis. The PPARÉ£ promoter reduced methylation significantly following zearalenone exposure. Taken together, the epigenetic mechanisms of DNA methylation and histone modifications may be key mechanisms in zearalenone toxicity. Furthermore, effects of zearalenone in metabolic pathways could be mediated by epigenetic modifications.

Photosensitive nanocarriers for specific delivery of cargo into cells.

Nanoencapsulation is a rapidly expanding technology to enclose cargo into inert material at the nanoscale size, which protects cargo from degradation, improves bioavailability and allows for controlled release. Encapsulation of drugs into functional nanocarriers enhances their specificity, targeting ability, efficiency, and effectiveness. Functionality may come from cell targeting biomolecules that direct nanocarriers to a specific cell or tissue. Delivery is usually mediated by diffusion and erosion mechanisms, but in some cases, this is not sufficient to reach the expected therapeutic effects. This work reports on the development of a new photoresponsive polymeric nanocarrier (PNc)-based nanobioconjugate (NBc) for specific photo-delivery of cargo into target cells. We readily synthesized the PNcs by modification of chitosan with ultraviolet (UV)-photosensitive azobenzene molecules, with Nile red and dofetilide as cargo models to prove the encapsulation/release concept. The PNcs were further functionalized with the cardiac targeting transmembrane peptide and efficiently internalized into cardiomyocytes, as a cell line model. Intracellular cargo-release was dramatically accelerated upon a very short UV-light irradiation time. Delivering cargo in a time-space controlled fashion by means of NBcs is a promising strategy to increase the intracellular cargo concentration, to decrease dose and cargo side effects, thereby improving the effectiveness of a therapeutic regime.