Overexpressing HepG2 Cells Research Articles

Gemcitabine enhances pharmacokinetic exposure of the major components of Danggui Buxue Decoction in rat via the promotion of intestinal permeability and down-regulation of CYP3A for combination treatment of non-small cell lung cancer

Context Danggui Buxue Decoction (DBD), a traditional Chinese medicine formula, has the potential to enhance the antitumor effect of gemcitabine in non-small cell lung cancer (NSCLC) treatment by increasing gemcitabine’s active metabolites. However, whether gemcitabine affects the pharmacokinetics of DBD’s major components remains unclear. Objective This study evaluates the herb–drug interaction between DBD’s major components and gemcitabine and validates the underlying pharmacokinetic mechanism. Materials and methods The pharmacokinetics of 3.6 g/kg DBD with and without a single-dose administration of 50 mg/kg gemcitabine was investigated in Sprague-Dawley rats. The effects of gemcitabine on intestinal permeability, hepatic microsomal enzymes in rat tissues, and CYP3A overexpressing HepG2 cells were determined using western blot analysis. Results The combination of gemcitabine significantly altered the pharmacokinetic profiles of DBD’s major components in rats. The C max and AUC of calycosin-7-O-β-d-glucoside notably increased through sodium-glucose transporter 1 (SGLT-1) expression promotion. The AUC of ligustilide and ferulic acid was also significantly elevated with the elimination half-life (t 1/2) prolonged by 2.4-fold and 7.8-fold, respectively, by down-regulating hepatic CYP3A, tight junction proteins zonula occludens-1 (ZO-1) and occludin expression. Discussion and conclusions Gemcitabine could modulate the pharmacokinetics of DBD’s major components by increasing intestinal permeability, enhancing transporter expression, and down-regulating CYP3A. These findings provide critical information for clinical research on DBD as an adjuvant for NSCLC with gemcitabine and help make potential dosage adjustments more scientifically and rationally.

Mitogen activated protein kinase phosphatase 5 alleviates liver ischemia–reperfusion injury by inhibiting TAK1/JNK/p38 pathway

Mitogen activated protein kinase phosphatase 5 (MKP5) is a member of the MKP family and has been implicated in diverse biological and pathological conditions. However, it is unknown what role MKP5 plays in liver ischemia/reperfusion (I/R) injury. In the present study, we used MKP5 global knockout (KO) and MKP5 overexpressing mice to establish a liver I/R injury model in vivo, and MKP5 knockdown or MKP5 overexpressing HepG2 cells to establish a hypoxia-reoxygenation (H/R) model in vitro. In this study we demonstrated that protein expression of MKP5 was significantly downregulated in liver tissue of mice after I/R injury, and HepG2 cells subjected to H/R injury. MKP5 KO or knockdown significantly increased liver injury, as demonstrated by elevated serum transaminases, hepatocyte necrosis, infiltrating inflammatory cells, secretion of pro-inflammatory cytokines, apoptosis, oxidative stress. Conversely, MKP5 overexpression significantly attenuated liver and cell injury. Furthermore, we showed that MKP5 exerted its protective effect by inhibiting c-Jun N-terminal kinase (JNK)/p38 activity, and its action was dependent on Transforming growth factor-β-activated kinase 1 (TAK1) activity. According to our results, MKP5 inhibited the TAK1/JNK/p38 pathway to protect liver from I/R injury. Our study identifies a novel target for the diagnosis and treatment of liver I/R injury.

Bee venom-loaded EGFR-targeting peptide-coupled chitosan nanoparticles for effective therapy of hepatocellular carcinoma by inhibiting EGFR-mediated MEK/ERK pathway.

Hepatocellular carcinoma (HCC) is one of the world's most risky diseases due to the lack of clear and cost-effective therapeutic targets. Currently, the toxicity of conventional chemotherapeutic medications and the development of multidrug resistance is driving research into targeted therapies. The nano-biomedical field's potential for developing an effective therapeutic nano-sized drug delivery system is viewed as a significant pharmaceutical trend for the encapsulation and release of numerous anticancer therapies. In this regard, current research is centered on the creation of biodegradable chitosan nanoparticles (CSNPs) for the selective and sustained release of bee venom into liver cancer cells. Furthermore, surface modification with polyethylene glycol (PEG) and GE11 peptide-conjugated bee venom-CSNPs allows for the targeting of EGFR-overexpressed liver cancer cells. A series of in vitro and in vivo cellular analyses were used to investigate the antitumor effects and mechanisms of targeted bee venom-CSNPs. Targeted bee venom-CSNPs, in particular, were found to have higher cytotoxicity against HepG2 cells than SMMC-7721 cells, as well as stronger cellular uptake and a substantial reduction in cell migration, leading to improved cancer suppression. It also promotes cancer cell death in EGFR overexpressed HepG2 cells by boosting reactive oxygen species, activating mitochondria-dependent pathways, inhibiting EGFR-stimulated MEK/ERK pathway, and elevating p38-MAPK in comparison to native bee venom. In hepatocellular carcinoma (HCC)-induced mice, it has anti-cancer properties against tumor tissue. It also improved liver function and architecture without causing any noticeable toxic side effects, as well as inhibiting tumor growth by activating the apoptotic pathway. The design of this cancer-targeted nanoparticle establishes GE11-bee venom-CSNPs as a potential chemotherapeutic treatment for EGFR over-expressed malignancies. Finally, our work elucidates the molecular mechanism underlying the anticancer selectivity of targeted bee venom-CSNPs and outlines therapeutic strategies to target liver cancer.

HIRA Supports Hepatitis B Virus Minichromosome Establishment and Transcriptional Activity in Infected Hepatocytes.

Background & AimsUpon hepatitis B virus (HBV) infection, partially double-stranded viral DNA converts into a covalently closed circular chromatinized episomal structure (cccDNA). This form represents the long-lived genomic reservoir responsible for viral persistence in the infected liver. Although the involvement of host cell DNA damage response in cccDNA formation has been established, this work investigated the yet-to-be-identified histone dynamics on cccDNA during early phases of infection in human hepatocytes.MethodsDetailed studies of host chromatin-associated factors were performed in cell culture models of natural infection (ie, Na+-taurocholate cotransporting polypeptide (NTCP)-overexpressing HepG2 cells, HepG2hNTCP) and primary human hepatocytes infected with HBV, by cccDNA-specific chromatin immunoprecipitation and loss-of-function experiments during early kinetics of viral minichromosome establishment and onset of viral transcription.ResultsOur results show that cccDNA formation requires the deposition of the histone variant H3.3 via the histone regulator A (HIRA)-dependent pathway. This occurs simultaneously with repair of the cccDNA precursor and independently from de novo viral protein expression. Moreover, H3.3 in its S31 phosphorylated form appears to be the preferential H3 variant found on transcriptionally active cccDNA in infected cultured cells and human livers. HIRA depletion after cccDNA pool establishment showed that HIRA recruitment is required for viral transcription and RNA production.ConclusionsAltogether, we show a crucial role for HIRA in the interplay between HBV genome and host cellular machinery to ensure the formation and active transcription of the viral minichromosome in infected hepatocytes.

A branched small molecule-drug conjugate nanomedicine strategy for the targeted HCC chemotherapy

Off-target toxicity is one of the main challenges faced by anticancer chemotherapeutics. For tumor targeted and precision chemotherapy, we take the advantages of the ligand directed tumor active targeting of small molecule drug conjugates (SMDCs) and the passive tumor targeting of nanoparticles via the enhanced penetration and retention (EPR) effects, put forward a branched small molecule drug conjugate (BSMDC) nanomedicine design concept. In a proof of concept, we used pentaerythritol as the branched moiety, galactosamine (GalN) as the hepatocellular carcinoma (HCC) directing ligands, PTX as a payload, and a stearoyl moiety as the amphiphilic property adjusting group, designed and synthesized BSMDC 1 and prepared its NPs. In cellular level, the BSMDC 1 NPs targeted asialoglycoprotein receptor (ASGPR)-overexpressing HepG2 cells, were effectively taken up in the cells and released in tumor microenvironments, inhibited the HepG2 cell proliferation, arrested HepG2 cell in G2/M phase and induced tumor cell apoptosis. In HepG2 xenograft nude mice, the BSMDC 1 NPs were high specific to target the tumor and demonstrated a higher antitumor efficiency than BSMDC 1, having no apparent influences on mice body weights and major organs, supporting our BSMDC nanomedicine design concept. Therefore, this new strategy may find applications for cancer targeted and precision chemotherapy.

A hepatocyte-specific fluorescent probe for imaging endogenous carbon monoxide release in vitro and in vivo

A novel single-component fluorescent probe HCP using N-acetylgalactosamine (GalNAc) as the hepatocyte-specific moiety and 3-nitrophthalimide as the fluorescent reactive unit was designed and synthesized to specifically detect hepatocellular carbon monoxide (CO) in vitro and in vivo. The spectral experimental results indicate that HCP displayed high selectivity to CO and could be used to detect trace CO in an aqueous solution with a prominent fluorescent response (more than 80 times). Bioimaging results show that HCP could specifically transport to asialoglycoprotein receptor overexpressed HepG2 cells and could image endogenous CO release in HepG2 cells and the liver of zebrafish in situ during acute liver injury.

Characterization of cytochrome P450s (CYP)-overexpressing HepG2 cells for assessing drug and chemical-induced liver toxicity

Hepatic metabolism catalyzed by the cytochrome P450 (CYP) superfamily affects liver toxicity associated with exposures to natural compounds and xenobiotic agents. Previously we generated a battery of HepG2-derived stable cell lines that individually express 14 CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7). In this study, we comprehensively characterized each cell line for its CYP expression and enzyme activity. Specifically, we measured the mRNA expression, protein expression, and metabolite formation. Using CYP3A4, 2D6, and 2C9-overexpressing cells as representatives, we examined the stability of these cells in long-term cultures for up to 10 passages. The results showed that CYPs can be stably overexpressed for up to 10 cell culture passages without losing their activities. The robustness of responses to stimuli among the cells at different passages was also investigated in CYP3A4-overexpressing cells and the response to amiodarone and dronedarone showed no difference between the cells at the passage 2 and 10. Moreover, the mRNA expression level of most CYPs was higher in CYP-overexpressing HepG2 cells than that in HepaRG cells and primary human hepatocytes. This study confirmed the stability of CYP-overexpressing HepG2 cell lines and provided useful information for a broader use of these cells in pharmacologic and toxicologic research.

A β-d-galactose-guided fluorescent probe for selectively bioimaging endogenous formaldehyde in living HepG-2 cells

A fluorescent probe NFP-G, with β-d-galactose as the targeted unit and hydrazine as the recognition group for formaldehyde (FA), was designed and synthesized for specifically detecting endogenous FA in HepG-2 cells via asialoglycoprotein receptor-mediated endocytosis. The experimental results show that probes NFP-G/NFP-A exhibited highly fluorescent responses toward FA, which could be ascribed to the restriction of the photoinduced electron transfer (PET) process by the formation of hydrazone. Good selectivity and competition of NFP-G toward FA over potentially interfering species ensure it an appropriate candidate for FA detection. The sensing mechanism of the probes toward FA was studied by theoretical calculation, high performance liquid chromatography and mass spectrum. The confocal bioimaging results reveal that NFP-G could selectively visualize the endogenous FA in asialoglycoprotein receptor overexpressed HepG-2 cells over other cells. Pretreated with β-galactosidase made both asialoglycoprotein receptor positive and negative cells display strong green fluorescence, revealing β-d-galactose is essential in targeting HepG-2 cells.

NFAT2 overexpression suppresses the malignancy of hepatocellular carcinoma through inducing Egr2 expression

BackgroundNuclear factor of activated T cells 2 (NFAT2) has been reported to regulate the development and malignancy of few tumors. In this study, we aimed to explore the effect of NFAT2 expression on cell fate of HepG2 cell and its potential mechanisms.MethodsFirstly, the pcDNA3.1-NFAT2 plasmid was transfected into HepG2 cells to construct NFAT2 overexpressed HepG2 cells. Then, the chemical count kit-8 cell viability assay, Annexin V-FITC apoptosis detection, EdU labeling proliferation detection, transwell and wound healing experiments were performed. The expression of Egr2 and FasL, and the phosphorylation of AKT and ERK, after ionomycin and PMA co-stimulation, was detected, while the Ca2+ mobilization stimulated by K+ solution was determined. At last, the mRNA and protein expression of NFAT2, Egr2, FasL, COX-2 and c-myc in carcinoma and adjacent tissues was investigated.ResultsThe NFAT2 overexpression suppressed the cell viability, invasion and migration capabilities, and promoted apoptosis of HepG2 cells. NFAT2 overexpression induced the expression of Egr2 and FasL and suppressed the phosphorylation of AKT and ERK. The sensitivity and Ca2+ mobilization of HepG2 cells was also inhibited by NFAT2 overexpression. Compared with adjacent tissues, the carcinoma tissues expressed less NFAT2, Egr2, FasL and more COX-2 and c-myc.ConclusionThe current study firstly suggested that NFAT2 suppressed the aggression and malignancy of HepG2 cells through inducing the expression of Egr2. The absence of NFAT2 and Egr2 in carcinoma tissues reminded us that NFAT2 may be a promising therapeutic target for hepatocellular carcinoma treatment.

Apoptosis contributes to the cytotoxicity induced by amodiaquine and its major metabolite N-desethylamodiaquine in hepatic cells

Amodiaquine (ADQ), an antimalarial drug used in endemic areas, has been reported to be associated with liver toxicity; however, the mechanism underlying its hepatoxicity remains unclear. In this study, we examined the cytotoxicity of ADQ and its major metabolite N-desethylamodiaquine (NADQ) and the effect of cytochrome P450 (CYP)-mediated metabolism on ADQ-induced cytotoxicity. After a 48-h treatment, ADQ and NADQ caused cytotoxicity and induced apoptosis in HepG2 cells; NADQ was slightly more toxic than ADQ. ADQ treatment decreased the levels of anti-apoptotic Bcl-2 family proteins, which was accompanied by an increase in the levels of pro-apoptotic Bcl-2 family proteins, indicating that ADQ-induced apoptosis was mediated by the Bcl-2 family. NADQ treatment markedly increased the phosphorylation of JNK, extracellular signal-regulated kinase (ERK1/2), and p38, indicating that NADQ-induced apoptosis was mediated by MAPK signaling pathways. Metabolic studies using microsomes obtained from HepG2 cell lines overexpressing human CYPs demonstrated that CYP1A1, 2C8, and 3A4 were the major enzymes that metabolized ADQ to NADQ and that CYP1A2, 1B1, 2C19, and 3A5 also metabolized ADQ, but to a lesser extent. The cytotoxicity of ADQ was increased in CYP2C8 and 3A4 overexpressing HepG2 cells compared to HepG2/CYP vector cells, confirming that NADQ was more toxic than ADQ. Moreover, treatment of CYP2C8 and 3A4 overexpressing HepG2 cells with ADQ increased the phosphorylation of JNK, ERK1/2, and p38, but not the expression of Bcl-2 family proteins. Our findings indicate that ADQ and its major metabolite NADQ induce apoptosis, which is mediated by members of the Bcl-2 family and the activation of MAPK signaling pathways, respectively.

Endothelial lipase increases antioxidative capacity of high-density lipoprotein

Endothelial lipase (EL) is a strong determinant of structural and functional properties of high-density lipoprotein (HDL). We examined whether the antioxidative capacity of HDL is affected by EL. EL-modified HDL (EL-HDL) and control EV-HDL were generated by incubation of HDL with EL- overexpressing or control HepG2 cells. As determined by native gradient gel electrophoresis, electron microscopy, and small-angle X-ray scattering EL-HDL is smaller than EV-HDL. Mass spectrometry revealed an enrichment of EL-HDL with lipolytic products and depletion of phospholipids and triacylglycerol. Kinetics of conjugated diene formation and HPLC-based malondialdehyde quantification revealed that EL-HDL exhibited a significantly higher resistance to copper ion-induced oxidation and a significantly higher capacity to protect low-density lipoprotein (LDL) from copper ion-induced oxidation when compared to EV-HDL. Depletion of the lipolytic products from EL-HDL abolished the capacity of EL-HDL to protect LDL from copper ion-induced oxidation, which could be partially restored by lysophosphatidylcholine enrichment. Proteomics of HDL incubated with oxidized LDL revealed significantly higher levels of methionine 136 sulfoxide in EL-HDL compared to EV-HDL. Chloramine T (oxidizes methionines and modifies free thiols), diminished the difference between EL-HDL and EV-HDL regarding the capacity to protect LDL from oxidation. In absence of LDL small EV-HDL and EL-HDL exhibited higher resistance to copper ion-induced oxidation when compared to respective large particles. In conclusion, the augmented antioxidative capacity of EL-HDL is primarily determined by the enrichment of HDL with EL-generated lipolytic products and to a lesser extent by the decreased HDL particle size and the increased activity of chloramine T-sensitive mechanisms.

1885-P: Caveolin1 Attenuates Palmitic Acid-Induced Hepatic Steatosis through Upregulating PGC1α-sirt3 Axis

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. Caveolae are flask-shaped invaginations of the plasma membrane enriched with cholesterol and phospholipid. Caveolin1 is the resident structural protein of caveolae and has previously been reported to involve in hepatic steatosis and NAFLD, while the mechanism remains elusive. Here we examined the effects of caveolin1 on palmitic acid (PA)-induced hepatic steatosis. Stable caveolin1 overexpressing HepG2 cells were developed by transfection with lentiviral particles containing human CAV1 protein (Lenti-CAV1). Lenti-Control served as a negative control. Compared with BSA treatment, PA treatment of HepG2 cells for 24 hours increased the lipid droplets and TG level (P<0.01). Compared with negative control, overexpression of caveolin1 attenuated the accumulation of lipid droplets and triglyceride content in HepG2 cells (P<0.05). At the same time, quantitative real-time PCR revealed that the mRNA expression of PGC1α and sirt3 were increased in caveolin1 overexpressing cells (P<0.05). In conclusion, caveolin1 may improve PA-induced hepatic steatosis through up-regulating PGC1α-sirt3 axis. Disclosure C. Lin: None. W. Zeng: None. S. Lin: None. K. Liu: None. L. Zeng: None.

ASPP2 enhances chemotherapeutic sensitivity through the down-regulation of XIAP expression in a p53 independent manner in hepatocellular carcinoma

Apoptosis stimulated protein of p53-2 (ASPP2) induces the transcription of p53-targeted genes to stimulates its pro-apoptosis function. The poor chemotherapeutic sensitivity is associated with the decreased ASPP2 expression in many human cancers. Here, multiple genes real-time RT-PCR array and western blotting analysis show that ASPP2 suppress the expression of X-linked inhibitor of apoptosis protein (XIAP), determinant of chemoresistance in cancer, in hepatocellular carcinoma (HCC) in a p53-independent manner. Further experiments with ASPP2-rAd and ASPP2-Lv confirmed that ASPP2 enhanced sensitivity of sorafenib to HCC via suppressing XIAP expression. XIAP mainly found on the cytoplasm and perinuclear areas of ASPP2 over-expressed HepG2 cells, while both cytoplasm and nucleus in ASPP2 shut down HepG2 cells. The association of poor sensitivity of sorafenib and XIAP expression was also found both in ASPP2 shut down and overexpress mice, where liver tissue with decreased or increased ASPP2 displayed less or more apoptosis, respectively. Finally, ASPP2 and XIAP expression analyzed in 43 hepatocellular carcinoma tumors and 44 adjacent normal tissues from 38 hepatocellular carcinoma patients for fully understand their expression within HCC patients. Compared with the tumor tissues, ASPP2 mRNA levels were increased, and XIAP levels decreased in the adjacent normal tissues. Taken together, XIAP suppressed ASPP2 increased tumor sensitivity to chemotherapy in a p53-independent manner, which was associated with chemotherapy resistance, suggesting that p53 activation and XIAP suppression were two independent ways that ASPP2 enhance the sensitivity of chemotherapy.

Galactose Multivalency Effect on the Cell Uptake Mechanism of Bioconjugated Nanoparticles

Galactose functionalized nanomaterials are commonly used for specific targeting/delivery of cancer cells, as galactose receptors are overexpressed in certain types of cancer cells. However, the role of galactose multivalency on cellular interaction, cell uptake mechanism, and subcellular targeting are largely unexplored. Here we show that the receptor mediated cellular internalization of galactose terminated nanoparticles depends on galactose multivalency. We have synthesized galactose functionalized multivalent quantum dots (QDs) of 15–20 nm hydrodynamic size with the average numbers of galactose per QD of 25, 50, and 80 [designated as QD(gal)25, QD(gal)50, and QD(gal)80] and investigated their uptake mechanism in galactose receptor overexpressed HepG2 cells. We found three distinct effects of galactose multivalency on the nanoparticle uptake mechanism. First, cellular interaction and uptake kinetics of nanoparticles increase with increasing galactose multivalency. Second, the cell uptake mechanism shift...

Facile fabrication of hypericin-entrapped glyconanoparticles for targeted photodynamic therapy.

BackgroundPhotodynamic therapy is a safe, noninvasive modality for cancer therapy, in which the photosensitizer (PS) is a crucial component. Hypericin (Hy) is a promising PS; however, its clinical application is significantly limited by its poor hydrophilicity.Materials and methodsTo overcome the clinical application limitation of Hy, a novel strategy is developed here by entrapping Hy into polydopamine (PDA) film formed on the surface of magnetic iron oxide nanoparticles (MNPs) through the self-polymerization of dopamine under alkaline condition. The amount of Hy in the Hy-entrapped PDA–MNP composite nanoparticles (denoted as PHMs) was measured by spectrophotometry. Furthermore, lactose, as the targeting ligand to asialoglycoprotein receptors, was conjugated to the surface of the PHMs by taking advantage of the spontaneous reaction of PDA with amino groups.ResultsSpectrophotometry analysis revealed that the amount of Hy in the PHMs was 72 μmol g−1 PHMs. The fabricated Hy-entrapped glyconanoparticle (Lac-PHM) exhibited excellent water dispersibility, stability, and selectivity for asialoglycoprotein receptors overexpressing HepG2 cells. Atomic absorption spectroscopy analysis showed that the amount of the Lac-PHMs taken in HepG2 cells was 2.1-fold higher than that of the triethylene glycol-modified PHMs. The results of intracellular reactive oxygen species generation detection, cytotoxicity study, and apoptosis detection indicated that the Lac-PHMs had a satisfying photodynamic effect to HepG2 cells.ConclusionThe strategy developed in this work offers great potential for delivery of a variety of hydrophobic PSs.

Multifunctional self-assembled micelles of galactosamine-hyaluronic acid-vitamin E succinate for targeting delivery of norcantharidin to hepatic carcinoma

A polymer of Galactosamine-hyaluronic acid-Vitamin E succinate (Gal-HA-VES) was designed to prepare multifunctional self-assembled micelles for delivery of Norcantharidin (NCTD) to Hepatic carcinoma. NCTD/Gal-HA-VES showed higher cytotoxicity toward CD44-overexpressing MCF-7 cells, MCF-7/Adr cells and ASGP-R overexpressing HepG2 cells, consistent with the enhanced cellular uptake in the selected cell models, indicating Gal-HA-VES micelles were taken up in MCF-7 and HepG2 cells by CD44 and ASGPR receptor mediated endocytosis, respectively. Moreover, the accumulation of Rhodamine 123 demonstrated that Gal-HA-VES has the same action of TPGS as a P-glycoprotein inhibitor blocked drug efflux-related MDR mechanism in resistant MCF-7/Adr cells. The Cell apoptosis assays indicated that NCTD/Gal-HA-VES were more effective in triggering apoptosis, compared with free NCTD or NCTD/HA-VES groups. In vivo study demonstrated that NCTD/Gal-HA-VES group exhibited enhanced tumor targeting and antitumor activity with lower systemic toxicity. Hence NCTD/Gal-HA-VES micelles system can achieve significant tumor targeting and effective treatment of hepatic carcinoma.

Acid-degradable lactobionic acid-modified soy protein nanogels crosslinked by ortho ester linkage for efficient antitumor in vivo

It remains a crucial challenge to achieve efficient cellular uptake and intracellular drug release in tumor cells for the nanoscale drug delivery systems. Herein, acid-degradable nanogels were prepared by cross-linking methacrylated soy protein with an acid-labile ortho ester cross-linker (NG1), and then modified with lactobionic acid (LA) to give tumor-targeted nanogels (NG2). Both NG1 and NG2 displayed excellent stability in neutral environment, while showed pH-triggered degradation behaviors under mildly acidic conditions resulting from the breakage of ortho ester bonds. Doxorubicin (DOX) was successfully loaded into nanogels, which exhibited an accelerated release at low pH. In vitro cell studies demonstrated that LA-modified nanogels could effectively improve cellular internalization, show higher cytotoxicity and apoptosis toward asialoglycoprotein receptor (ASGPR) over-expressed HepG2 cells. In vivo antitumor experimentproved that LA modification could significantly enhance the tumor-targeting ability of nanogels and increase DOX concentration in tumor site, leading to better therapeutic efficacy. Histological analysis further demonstrated that soy protein-based nanogels did not cause any damage to normal organs. Overall, these pH-sensitive and tumor-targeting soy protein-based nanogels can be potential drug carriers for efficient tumor treatment.

The histidine triad nucleotide-binding protein 2 (HINT-2) positively regulates hepatocellular energy metabolism.

The histidine triad nucleotide-binding protein 2 (HINT-2) is a mitochondrial adenosine phosphoramidase expressed in hepatocytes. The phenotype of Hint2 knockout ( Hint2-/-) mice includes progressive hepatic steatosis and lysine hyperacetylation of mitochondrial proteins, which are features of respiratory chain malfunctions. We postulated that the absence of HINT-2 induces a defect in mitochondria bioenergetics. Isolated Hint2-/- hepatocytes produced less ATP and generated a lower mitochondrial membrane potential than did Hint2+/+ hepatocytes. In extracellular flux analyses with glucose, the basal, ATP-linked, and maximum oxygen consumption rates (OCRs) were decreased in Hint2-/- hepatocytes and in HepG2 cells lacking HINT-2. Conversely, in HINT-2 overexpressing SNU-449 and HepG2 cells, the basal, ATP-linked, and maximum OCRs were increased. Similarly, with palmitate, basal and maximum OCRs were decreased in Hint2-/- hepatocytes, but they were increased in HINT-2 overexpressing HepG2 cells. When assayed with radiolabeled substrate, palmitate oxidation was reduced by 25% in Hint2-/- mitochondria. In respirometry assays, complex I- and II-driven, coupled and uncoupled respirations and complex IV KCN-sensitive respiration were reduced in Hint2-/- mitochondria. Furthermore, HINT-2 associated with cardiolipin and glucose-regulated protein 75 kDa. Our study shows decreased electron transfer and oxidative phosphorylation capacity in the absence of HINT-2. The bioenergetics deficit accumulated over time in hepatocytes lacking HINT-2 likely leads to the secondary outcome of steatosis.-Rajasekaran, R., Felser, A., Nuoffer, J.-M., Dufour, J.-F., St-Pierre, M. V. The histidine triad nucleotide-binding protein 2 (HINT-2) positively regulates hepatocellular energy metabolism.

Low-density lipoprotein decorated silica nanoparticles co-delivering sorafenib and doxorubicin for effective treatment of hepatocellular carcinoma

Combinational therapy is usually considered as a preferable approach for effective cancer therapy. Especially, combinational chemotherapies targeting different molecular targets are of particular interest due to its high flexibility as well as efficiency. In our study, the surface of silica nanoparticles (SLN) was modified with low-density lipoprotein (LDL) to construct platform (LDL-SLN) capable of specifically targeting low-density lipoprotein receptors (LDLRs) that overexpressing in hepatocellular carcinoma (HCC). In addition, the versatile drug loading capacity of LDL-SLN was employed to fabricate a preferable drug delivery system to co-deliver sorafenib (Sor) and doxorubicin (Dox) for combinational chemotherapy of HCC. Our results revealed that the LDL-SLN/Sor/Dox nanoparticles with size around 100 nm showed preferable stability in physiological environments. Moreover, the LDL-SLN/Sor/Dox could target LDLR overexpressed HepG2 cells. More importantly, both in vitro and in vivo experiments demonstrated that the LDL-SLN/Sor/Dox exerted elevated antitumor efficacy compared to Sor or Dox alone, which indicated that LDL-SLN/Sor/Dox could be a powerful tool for effective combinational chemotherapy of HCC.

Morin impedes Yap nuclear translocation and fosters apoptosis through suppression of Wnt/β-catenin and NF-κB signaling in Mst1 overexpressed HepG2 cells

Recent clinical and experimental evidences strongly acclaim Yes-associated protein (Yap), a key oncogenic driver in liver carcinogenesis, as a therapeutic target. Of the known multiple schemes to inhibit Yap activity, activation of Mammalian Sterile 20-like Kinase 1 (Mst1), an upstream regulator of Yap, appears to be a promising one. In this study, we hypothesize that morin, a bioflavonoid, mediates its anti-cancer effect through the activation of Mst1/hippo signaling in liver cancer cells. To test this hypothesis, both full length Mst1 (F-Mst1) and kinase active N-terminal Mst1 (N-Mst1)-overexpressed HepG2 cells were used. Exposure of F-Mst1 overexpressed HepG2 cells to morin activated Mst1 by caspase-3 cleavage and thereby inhibited Yap nuclear translocation and fostered apoptosis. Morin suppressed NF-κB p65 and Wnt/β-catenin signaling through Mst1 activation via cleavage and phosphorylation, leading to cell death. Annexin-V/PI staining further confirmed the induction of apoptosis in morin treated F-Mst1 overexpressed cells. The present study shows that morin targets cell survival molecules such as NF-κB p65 and β-catenin through activation of hippo signaling. Therefore, morin could be considered as a potential anti-cancer agent against liver cancer.