Normal Human Hepatic Cells Research Articles

Nicotine Promotes WRL68 Cells Proliferation Due to the Mutant p53 Gain-of-Function by Activating CDK6-p53-RS-PIN1-STAT1 Signaling Pathway.

The liver is an important organ with many tasks, such as dealing with drugs, alcohol and other toxins to remove them from the body. Nicotine is the more abundant component in cigarette smoking, which is first metabolized in liver and increases the risk of developing hepatocellular carcinoma (HCC). Also, genotoxic potential of nicotine has been extensively studied in vitro. However, the carcinogenic action of nicotine on the HCC needs to be elucidated. The current study demonstrated that chronic exposure to nicotine significantly promotes human normal fetal hepatic cell line (WRL68 cells) proliferation in a time- and concentration-dependent manner resulting from G0/G1-S-phase transition. Also remarkably, nicotine induced the level of p53 mutation at Ser249 (p53-RS). Note as well that the level of STAT1 protein was increased along with p53-RS owing to the prolonged half-life of STAT1. Furthermore, it is suggested that CDK6-dependent binding between phosphorylation of p53-RS at Ser249 and PIN1 by nicotine treatment leads to the nucleus translocation, followed by interacting with STAT1 and subsequent activation of STAT1 via the improvement of its stability, which is involved in cellular growth and colony formation after nicotine treatment. Simply put, these findings indicated that nicotine induces mutant p53 gain-of function (GOF), activating CDK6-p53-RS-PIN1-STAT1 signaling pathway and promoting cell proliferation, which could contribute to HCC for smokers.

Downregulation of miR-1225-5p is pivotal for proliferation, invasion, and migration of HCC cells through NFκB regulation.

BackgroundAs one of the most frequently seen malignancies, hepatocellular carcinoma (HCC) serves as the second largest contributor to malignancy‐specific mortality worldwide. MicroRNA‐1225‐5p (miR‐1225) exerts an essential impact on the growth and metastasis of many malignancies. However, the contribution of miR‐125 to HCC and the molecular mechanism of cancer cell viability and apoptosis are still unclear. We focused our research on exploring the function and molecular mechanism of miR‐1225 in regulating HCC cell growth, migration, and invasion.MaterialQuantitative PCR data showed that miR‐1225 expression was repressed in HCC cell lines and in the tissues of HCC patients, compared to that in normal human hepatic cells and tissues. Transfection of a miR‐1225 mimic inhibited cell viability and proliferation as indicated by CCK‐8 staining and MTT assay. Transwell invasion, wound healing assay, and Western blotting were performed to assess whether miR‐1225 repressed the metastasis and invasion of HCC cells, and decreased matrix metalloproteinase 9 (MMP9) expression. Further bioinformatic prediction and dual‐luciferase reporter assay suggested that miR‐1225 targeted the 3′‐UTR of NFκB p65.ResultsOverexpression of p65 protein counteracted the repressive impact of miR‐1225 on invasion, migration, and proliferation of HCC cells.ConclusionThis research provided new evidences that miR‐1225 inhibits the viability, migration, and invasion of HCC cells by downregulation of p65.

FOXA1 Promotes Cell Proliferation and Suppresses Apoptosis in HCC by Directly Regulating miR-212-3p/FOXA1/AGR2 Signaling Pathway.

BackgroundForkhead box protein A1 (FOXA1), acting as a transcriptional activator for liver-specific transcripts, plays a vital part in proliferation, apoptosis and cell cycle.MethodsThe mRNA expression of FOXA1 in 90 HCC tissues and matched adjacent non-tumor tissues was determined by qRT-PCR. The downstream and upstream regulators of FOXA1 were identified by bioinformatics analysis and experimental confirmation.ResultsWe found out that the expression of FOXA1 was obviously higher in hepatocellular carcinoma (HCC) tissues than that in matched non-tumor tissues. Similarly, FOXA1 is also highly expressed in HCC cell lines as compared with normal human hepatic cell line L02. Clinical association analysis indicated that high expression of FOXA1 was prominently correlated with high HBV level, large tumor size, high venous infiltration, high Edmondson–Steiner grading, and advanced tumor-node-metastasis tumor stage. Furthermore, the in vitro tests showed that ectopic expression of FOXA1 promoted HepG2 cell proliferation and suppressed apoptosis. In contrast, the downregulation of FOXA1 inhibited cell proliferation, and induced apoptosis in Hep3B cells. To investigate the functional mechanism of FOXA1, anterior gradient 2 (AGR2), an executor in proliferation and apoptosis, was identified as the direct target gene of FOXA1. Meanwhile, we also found the expression of FOXA1 could be inhibited by miR-212-3p, which working as a tumor suppressor downregulated in HCC.ConclusionWe revealed that FOXA1 exerted its biological function by regulating AGR2 expression, and its ectopic expression may be blamed for low expression of miR-212-3p.

Platinum nanoparticles induced genotoxicity and apoptotic activity in human normal and cancer hepatic cells via oxidative stress-mediated Bax/Bcl-2 and caspase-3 expression.

Platinum nanoparticles (PtNPs) attract much attention due to their excellent biocompatibility and catalytic properties, but their toxic effects on normal (CHANG) and cancerous (HuH-7) human liver cells are meagre. The cytotoxic and apoptotic effects of PtNPs (average size, 3 nm) were determined in CHANG and HuH-7 cells. After treating these cells were with PtNPs (10, 50, 100, 200, and 300 μg/mL) for 24 and 48 hours, we observed dose- and time-dependent cytotoxicity, as evaluated by using (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide, a tetrazole) (MTT) and neutral red uptake (NRU) assays. The production of reactive oxygen species (ROS) was increased in both cells after treatment with the above dose of PtNPs for 24 and 48 hours. Determination of morphological changes of cells, chromosome condensation, mitochondrial membrane potential, and caspase-3 assays showed that PtNPs induce cytotoxicity and apoptosis in CHANG and HuH-7 cells by altering the cell morphology and density, increasing cell population in apoptosis, and causing chromosome condensation. Furthermore, we have studied fragmentation of DNA using alkaline single cell gel electrophoresis and expression of apoptotic genes by real-time PCR (RT-PCR). The percentage of DNA fragmentation was more at 300 μg/mL for 48 hours in both cells, but slightly more fragmentation was found in HuH-7 relative to CHANG cells. Considering all of the above parameters, PtNPs elicited cytotoxicity on CHANG and HuH-7 cells by blocking cell proliferation and inducing apoptosis. Thus this study may be useful in in vitro laboratory studies using cell lines for screening the genotoxic and apoptotic potential of nanoparticles.

Antioxidant, α-amylase and α-glucosidase activity of various solvent fractions of I. obliquus and the preventive role of active fraction against H2 O2 induced damage in hepatic L02 cells as fungisome.

Inonotus obliquus is a traditional mushroom well known for its therapeutic value. In this study, various solvent fractions of I. obliquus were preliminarily screened for their antioxidant, α-amylase and α-glucosidase inhibition properties. To improve the drug delivery, the active fraction (ethyl acetate fraction) of I. obliquus was synthesized into fungisome (ethyl acetate phophotidyl choline complex, EAPC) and its physical parameters were assessed using Fourier transform infrared spectroscopy (FTIR), High performance liquid chromatography (HPLC), Scanning electron microscope (SEM), and ς potential analysis. Then normal human hepatic L02 cells was used to evaluate the cytotoxicity of EAPC. The results showed that EA fraction possesses significant free radical scavenging, α-amylase and α-glucosidase inhibition properties. FTIR, SEM, and HPLC analysis confirmed the fungisome formation. The particle size of EAPC was 102.80±0.42nm and the ς potential was -54.30±0.61mV. The percentage of drug entrapment efficiency was 97.13% and the drug release rates of EAPC in simulated gastric fluid and simulated intestinal fluid were 75.04±0.29% and 93.03±0.36%, respectively. EAPC was nontoxic to L02 cells, however it could selectively fight against the H2 O2 induced oxidative damage in L02 cells. This is the first study to provide scientific information to utilize the active fraction of I. obliquus as fungisome. PRACTICAL APPLICATIONS: Inonotus obliquus (IO) is a traditional medicinal fungus. The extracts of IO have obvious antioxidant and hypoglycemic activities. Ethyl acetate (EA) fraction of IO was encapsulated in liposomes to form EAPC. EAPC has a sustained-release effect. It has nontoxic to L02 cells and could protect L02 cells from oxidative damage caused by hydrogen peroxide. This study could provide new ideas for the treatment of diabetes.

Long Noncoding RNA LINC00460 Promotes Hepatocellular Carcinoma Progression via Regulation of miR-342-3p/AGR2 Axis.

BackgroundHepatocellular carcinoma (HCC) is the leading cause of cancer-related death worldwide. LINC00460, a novel long non-coding RNA (lncRNA), was recently confirmed as an oncogene in various cancers. However, the biological function and underlying mechanism of LINC00460 in HCC is largely obscure.MethodsFifty pairs of tumor tissue and adjacent normal tissues from HCC patients, as well as six HCC cell lines and a normal human hepatic epithelial cell line were subjected to qRT-PCR assay to evaluate the expression levels of LINC00460. CCK-8 assays were used to detect the proliferation of HCC cells. Transwell assay was used to measure the migration and invasion abilities of HCC cells. RNA pull-down and luciferase assays were performed to verify the direct interaction between LINC00460 and miR-342-3p. A xenograft model of HCC was established to validate the in vivo function of LINC00460 in HCC progression.ResultsWe firstly detected LINC00460 expression was significantly upregulated in both HCC tumor tissues and cell lines. The upregulation of LINC00460 was positively associated with HCC progression. Functionally, LINC00460 facilitated HCC cell proliferation, migration, and invasion capacities, which due to that LINC00460 could physically bind to and repress miR-342-3p to elevate the expression of AGR2.ConclusionOur data firstly reveal the clinical relevance, biological function, and regulatory mechanism of LINC00460 in HCC development. LINC00460 promotes HCC progression by elevating AGR2 expression via sponging miR-342-3p, providing a promising therapeutic target for HCC treatment.

Effects of Green Silver Nanoparticles on Apoptosis and Oxidative Stress in Normal and Cancerous Human Hepatic Cells in vitro.

IntroductionExtensive use of metallic nanomaterials in different areas of agriculture and commercial products induce significant harmful effects on human health and the environment. In the current study, we synthesized an eco-friendly approach silver nanoparticles (AgNPs) using root extracts of Beta vulgaris L.MethodsThe synthesized green silver nanoparticles (gAgNPs) were characterized by dynamic light scattering (DLS) and high-resolution transmission electron microscope (HR-TEM). The gAgNPs had a round shape and the mean size was 20−50 nm. The cytotoxic effects of gAgNPs were determined in human hepatic normal (CHANG) and cancer (HUH-7) cells by using tetrazolium salt (MTT) and lactate dehydrogenase (LDH) assays for 24 h.Results and DiscussionIt was clear from the observations of this experiment that higher concentrations of gAgNPs reduce cell viability. The production of reactive oxygen species (ROS) was evaluated by using DCFDA. The gAgNPs induced more ROS in the HuH-7 cells than in the CHANG cells. The fragmentation of DNA was evaluated by alkaline single-cell gel electrophoresis and the maximum DNA strand breakage was found at a higher concentration exposure of gAgNPs for 24 h. It is important to notice that the HuH-7 cells showed an increased sensitivity to gAgNPs than the CHANG cells. The apoptotic and necrotic effects of gAgNPs on both the cells were evaluated using annexin-V-FITC and propidium iodide staining. An increased count of apoptotic and necrotic cells was found following a higher concentration exposure of gAgNPs. The apoptotic protein expression in these cells due to gAgNPs exposure was determined using immunoblotting techniques and the level of Bcl2 was decreased. However, the expression of BAX and protein was increased in both cells.ConclusionTherefore, it can be concluded that higher concentrations of gAgNPs may induce significant cytotoxicity and cause DNA damage and apoptosis.

Oleanolic Acid Induces Apoptosis and Necrosis in Lo2 Cells

This study mainly focused on the evaluation of cytotoxicity of oleanolic acid on normal human hepatic cells. Normal human hepatic cells were exposed to different concentrations of oleanolic acid and the results demonstrated that oleanolic acid induced cell death in a concentration- and time-dependent manner in LO2 cells. Low concentration of oleanolic acid (40 μM) induced apoptosis. Cleavage of caspase 3/7/9 and decrease of mitochondrial membrane potential was detected in normal human hepatic cells. While high concentration of oleanolic acid (80 μM) caused necrosis without producing cleavage of caspase 3/7/9 or DNA fragmentation. Both flow cytometry and PI staining detected necrotic cells. In addition, both low and high concentrations of oleanolic acid had no effect on the generation of intracellular reactive oxygen species. Present data showed that oleanolic acid could induce apoptosis and necrosis in normal human hepatic cells. Oleanolic acid might be a potential hepatotoxin at high dosage.

Solvothermal Synthesis of Novel Magnetic Nickel Based Iron Oxide Nanocomposites for Selective Capture of Global- and Mono-Phosphopeptides.

A facile solvothermal method was developed for synthesis of magnetic nickel-based iron oxide nanocomposites (MNFOs) with different ratios of Ni2+ to Fe3+ for different reaction time. Two factors including dosage of Ni source and length of reaction were investigated for influence on the morphology and composition of MNFOs, as well as their distinct selectivity for different phosphopeptides. After thorough characterization, the possible formation mechanism of MNFOs was proposed. Very interestingly, MNFOs with Ni2+/Fe3+ ratios of 4:5 prepared for 8 h (MNFO-S) and for 24 h (MNFO-L) can selectively capture global- and monophosphopeptides at the fmol level with excellent enrichment performance. These two affinity probes have been exploited to isolate and enrich the phosphopeptides from human normal hepatic cells HL 7702 after exposure to atmospheric fine particulates (PM2.1), which revealed that the protein phosphorylation level was increased significantly in cells after stimulation by fine particulate matters. The findings could provide a new insight for the nickel-based affinity protocol to analyze the mutation of phosphopeptides during cellular signaling pathways in response to exogenous environment stimulation. Consequently, this present work proposed a promising strategy to isolate monophosphopeptides from global phosphopeptides for phosphoproteome research.

Cyclooxygenase-2 modulates ER-mitochondria crosstalk to mediate superparamagnetic iron oxide nanoparticles induced hepatotoxicity: an in vitro and in vivo study

Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are central microdomains of the ER that interact with mitochondria. MAMs provide an essential platform for crosstalk between the ER and mitochondria and play a critical role in the local transfer of calcium (Ca2+) to maintain cellular functions. Despite the potential uses of superparamagnetic iron oxide nanoparticles (SPIO-NPs) in biomedical applications, the hepatotoxicity of these nanoparticles (NPs) is not well characterized and little is known about the involvement of MAMs in ER-mitochondria crosstalk. We studied SPIO-NPs-associated hepatotoxicity in vitro and in vivo. In vitro, human normal hepatic L02 cells were exposed to SPIO-NPs (2.5, 7.5, and 12.5 μg/mL) for 6 h and SPIO-NPs (12.5 μg/mL) was found to induce apoptosis. In vivo, SPIO-NPs induced liver injury when mice were intravenously injected with 20 mg/kg body weight SPIO-NPs for 24 h. Based on both in vitro and in vivo studies, we found that the structure and Ca2+ transport function of MAMs were perturbated and an accumulation of cyclooxygenase-2 (COX-2) in MAMs fractions was increased upon treatment of SPIO-NPs. The interaction between COX-2 and the components of MAMs, in terms of IP3R-GRP75-VDAC1 complex, was also revealed. Furthermore, the role of COX-2 in SPIO-NPs-associated hepatotoxicity was investigated by modifying the expression of COX-2. We demonstrated that COX-2 increases the structural and functional ER-mitochondria coupling and enhances the efficacy of ER-mitochondria Ca2+ transfer through the MAMs, thus sensitizing hepatocytes to a mitochondrial Ca2+ overload-dependent apoptosis. Taken together, our findings link SPIO-NPs-triggered hepatotoxicity with ER-mitochondria Ca2+ crosstalk which is mediated by COX-2 and provide mechanistic insight into the impact of interorganelle ER-mitochondria communication on hepatic nanotoxicity.

Design, synthesis, in vitro and in vivo evaluation of novel pyrrolizine-based compounds with potential activity as cholinesterase inhibitors and anti-Alzheimer's agents

Novel series of pyrrolizine based compounds (4–6 and 9–11) were designed, synthesized and evaluated as potential anti-Alzheimer agents. Most of the tested compounds showed selectivity to hAChE over hBChE and effectively inhibited self–induced amyloid beta aggregation in vitro. Among these derivatives, compound 10 displayed high selectivity towards hAChE (Ki = 1.47 ± 0.63 μM for hAChE and Ki = 40.15 ± 3.31 μM for hBChE). However, compound 11 displayed dual inhibitory effect against hAChE and hBChE at submicromolar range (Ki = 0.40 ± 0.03 and 0.129 ± 0.009 μM, respectively). Kinetic studies of the new ligands showed competitive type inhibition for both hAChE and hBChE. Moreover, compounds 10 and 11 showed lower or comparable cytotoxicity to donepezil against human neuroblastoma (SH-SY5Y) and normal human hepatic (THLE2) cell lines. In vivo studies confirmed that both compounds were able to improve cognitive dysfunction of scopolamine-induced AD mice. Finally, molecular docking simulation of compounds 10 and 11 in hAChE active site showed good agreement with the obtained pharmaco-biological results.

The role of circular RNA hsa_circ_0085616 in proliferation and migration of hepatocellular carcinoma cells.

BackgroundCircRNAs involved in the development of many diseases have been recently identified. However, the possible role of circRNAs in human hepatocellular carcinoma (HCC) remains to be investigated.ObjectiveThis study aimed to identify the expression of hsa_circ_0085616 in different HCC cell lines and its function in HCC tumorigenesis.MethodsThe expression of hsa_circ_0085616 was first measured in 68 pairs of HCC tissues and matched normal adjacent tissues. Further analysis was performed in different HCC cell lines and human normal hepatic cell lines. Moreover, we down- or upregulated its expression by cell transfection in vitro. Cell proliferation, migration, invasion and apoptosis were measured, and proliferation-related signaling pathway proteins were also analyzed.ResultsWe found that hsa_circ_0085616 was highly expressed in all HCC cell lines compared to the normal liver cell line. Upregulation or downregulation of hsa_circ_0085616 expression could strengthen or weaken the proliferative ability of HCC cells in vitro. Moreover, the protein levels of β-catenin, p-ERK, and p-AKT, which play important roles in the typical proliferation pathway, were also affected by the expression of hsa_circ_0085616.ConclusionOur study indicated that hsa_circ_0085616 might be a potential biomarker and a new therapeutic target for HCC.

MiR-451a suppresses cell proliferation, metastasis and EMT via targeting YWHAZ in hepatocellular carcinoma.

MicroRNAs (miRNAs) have been identified to participate in the progression of hepatocellular carcinoma (HCC). However, the function of miR-451a in HCC remains unknown. The aim of this study was to determine the function of miR-451a by construction of several experiments in HCC tissues and cells. The expression level of miR-451a in 69 paired of HCC and adjacent normal tissue samples was detected using quantitative Real-time polymerase chain reaction (qRT-PCR). MiR-451a expression in HCC derived cell lines was detected as well. By transfecting with miR-451a mimics or inhibitor, the expression level of miR-451a in HepG2 or Huh-7 cells was up- or down-regulated. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and colony formation analysis were employed to evaluate the changes of cell proliferation. Cell migration and invasion abilities were measured via transwell assay. Meanwhile, the underlying mechanism of miR-451a in HCC was demonstrated and verified by dual-luciferase assay and Western blotting, respectively. Rescue experiments were used to identify the downstream molecule of miR-451a in HCC. MiR-451a expression in HCC tissue samples was significantly lower than that of adjacent normal samples. Meanwhile, the level of miR-451a in HCC cell lines was significantly down-regulated when compared with normal human hepatic cell line LO2. MTT assay and colony formation analysis showed that over-expressed miR-451a remarkably inhibited proliferation of HepG2 cells, whereas down-regulated miR-451a promoted growth of Huh-7 cells. Transwell results indicated that up-regulation of miR-451a significantly decreased HCC cell invasion and migration, while down-regulation remarkably increased cell metastasis. Furthermore, YWHAZ was identified as a direct target for miR-451a in HCC cells. The expression level of miR-451a was decreased in HCC tissues and cell lines. Moreover, miR-451a inhibited the proliferation, invasion and migration of HCC cells via targeting YWHAZ. Our findings indicated that miR-451a could serve as a novel target for HCC diagnosis and biological therapy.

465 – Comparative Analysis of Gene Expression in Primary Human Normal and Alcohol-Injured Hepatic Stellate Cells Identifies Novel Targets for Anti-Fibrotic Therapy Authors

465 – Comparative Analysis of Gene Expression in Primary Human Normal and Alcohol-Injured Hepatic Stellate Cells Identifies Novel Targets for Anti-Fibrotic Therapy Authors

MiR-1-3p suppresses proliferation of hepatocellular carcinoma through targeting SOX9.

BackgroundLiver cancer was the fourth leading cause of cancer-related death in 2015. Hepatocellular carcinoma (HCC) is the most common type of liver cancer. miR-1-3p plays important roles in cancer, including prostate, bladder, lung cancer, and colorectal carcinoma. The function of miR-1-3p in HCC remains poorly understood.MethodsqRT-PCR was performed to detect the miR-1-3p expression in HCC cell lines (HCCLM3, Hep3B, Bel-7404, SMMC-7721) and the normal human hepatic cell line (LO2). HCCLM3 and Bel-7404 cells were transfected with miR-1-3p mimic or scramble control followed by water-soluble tetrazolium salt (WST-1) assay. Western bolt analysis was performed to determine the protein levels. TargetScan7.1 (http://www.targetscan.org/vert_71/) was used to predict the potential targets of miR-1-3p. SRY (sex determining region Y)-box 9 (SOX9), which has been previously shown to play an important role in HCC, was found to be a target of miR-1-3p. Luciferase reporter assay was used to explore the targeting of miR-1-3p on SOX9. For in vivo tumorigenesis assay, HCCLM3 cells with stable overexpression of miR-1-3p or control plasmid were injected subcutaneously into the flank of the SCID mice and animals were monitored for tumor growth.ResultsmiR-1-3p was significantly downregulated in HCC cell lines (HCCLM3, Hep3B, Bel-7404, and SMMC-7721) compared to normal human hepatic cell line (LO2). Overexpression of miR-1-3p significantly inhibited the proliferation and induced apoptosis in HCCLM3 and Bel-7474 cells. SOX9 was a direct target of miR-1-3p in HCC cells. Inhibition of SOX9 significantly inhibited the proliferation of HCCLM3 and Bel-7474 cells. In vivo, overexpression of miR-1-3p decreased tumor volume in a xenograft model.ConclusionThese results highlight the role of miR-1-3p in HCC. Overexpression of miR-1-3P inhibited the proliferation of HCC at least partly due to the regulation of SOX9. miR-1-3p may be a promising therapeutic candidate for HCC.

Artemisinin-indole and artemisinin-imidazole hybrids: Synthesis, cytotoxic evaluation and reversal effects on multidrug resistance in MCF-7/ADR cells

A series of artemisinin derivatives with MDR reversal activity were designed and synthesized. All hybrids were screened to anticancer activities against four human cancer cell lines (A549, MCF-7, HepG-2, MDA-MB-231) and normal human hepatic cell (L02) in vitro. Most of the new compounds showed higher anticancer activities than artemisinin, among which compounds 11a and 11c displayed superior potency with IC50 6.78 μM and 5.25 μM against MCF-7, respectively. The further research indicated that the most potent 11c induced cell cycle arrest at G2 phase in MCF-7. Additionally, compound 11c showed remarkable MDR reversal activity which reversed adriamycin against MCF-7/ADR cells with IC50 0.76 μM.

Titanium Dioxide Nanoparticles Enhance Leakiness and Drug Permeability in Primary Human Hepatic Sinusoidal Endothelial Cells.

Liver sinusoidal endothelial cells (LSECs) represent the permeable interface that segregates the blood compartment from the hepatic cells, regulating hepatic vascular tone and portal pressure amidst changes in the blood flow. In the presence of pathological conditions, phenotypic changes in LSECs contribute to the progression of chronic liver diseases, including the loss of endothelial permeability. Therefore, modulating LSECs offers a possible way to restore sinusoidal permeability and thereby improve hepatic recovery. Herein, we showed that titanium dioxide nanoparticles (TiO2 NPs) could induce transient leakiness in primary human hepatic sinusoidal endothelial cells (HHSECs). Interestingly, HHSECs exposed to these NPs exhibited reduced protein kinase B (Akt) phosphorylation, an important protein kinase which regulates cell attachment. Using a 3D co-culture system, we demonstrated that TiO2 NPs diminished the attachment of HHSECs onto normal human hepatic cell LO2. To further illustrate the significance of leakiness in liver sinusoids, we showed that NP-induced leakiness promoted Sunitinib transport across the HHSEC layer, resulting in increased drug uptake and efficacy. Hence, TiO2 NPs have the potential to modulate endothelial permeability within the specialized sinusoidal endothelium, especially during events of fibrosis and occlusion. This study highlighted the possible use of inorganic NPs as a novel strategy to promote drug delivery targeting the diseased liver.

Knockdown of FOXK1 suppresses liver cancer cell viability by inhibiting glycolysis

AimsAerobic glycolysis is a unique characteristic of cancer cells, and this key signature provides a target for therapeutic intervention of cancers. Forkhead box protein K1 (FOXK1) is a transcriptional factor that plays an oncogenic role in several types of cancers, including liver cancer. However, whether FOXK1 could affect glycolysis in cancer cells remains unknown. The aim of the present study was to evaluate the effect of FOXK1 on glycolysis in liver cancer cells. Main methodsThe expression of FOXK1 in four human liver cancer cell lines was evaluated using qRT-PCR and western blot. Cell viability was assessed using MTT and CCK-8 assays. Glucose consumption and lactate production of the cells were detected to reflect glycolysis. The expression of hexokinase 2 (HK2) was detected using qRT-PCR and western blot. Key findingsFOXK1 was up-regulated in liver cancer cells as compared to the normal human hepatic cell line. Knockdown of FOXK1 reduced cell viability and HK2 expression. FOXK1 knockdown decreased glucose consumption and lactate production in liver cancer cells. Furthermore, FOXK1 knockdown suppressed the activation of Akt/mTOR pathway. Inhibition of Akt/mTOR pathway reduced cell viability and glycolysis of liver cancer cells. SignificanceThese findings suggested that FOXK1 knockdown inhibited cell viability of liver cancer cells by regulating glycolysis through the Akt/mTOR pathway.

Effect of Meloxicam on invasion and migration of liver cancer SMMC-7721 cellS and itsmechanism

Objective To explore the effect and mechanism of Meloxicam on migration and invasion of SMMC-7721 cells. Methods Trypan blue dye exclusion assay was used to determine the cytotoxic effect of varying dosages of Mel on the HL-7702 normal human hepaticcells. The migration and invasion of SMMC-7721 cells were determined by wound scratch assay and Transwell™ assay respectively. The expression of cyclooxygenase-2 (COX-2), E-cadherin, N-cadherin, matrix metalloproteinase (MMP)-2 in SMMC-7721 cells treated by Mel were detected by Western blotting. Results 0-200 μmol/ml Mel were not significantly toxic to HL-7702 normal human hepaticcells, 400 μmol/ml Mel had significant cytotoxic to HL-7702 normal human hepaticcells (P=0.007). the abilities of invasion and migration of SMMC-7721 cells were inhibited by Mel. The expression of COX-2 and N-cadherin were decreased but E-cadherin increased and MMP-2 had no significant difference after Mel treatment (P=0.128). Conclusion Mel is less toxic to normal cells, also can inhibit the abilities of proliferation, migration and invasion of SMMC-7721 cells that might be related to regulate the expression of COX-2, N-cadherin and E-cadherin. Thus, the transformation process of epithelial-mesenchymal transition (EMT). Key words: Meloxicam; SMMC-7721 cells; Migration; Invasion

Polyclonal antibody preparation against candidate tumour suppressor protein MIP for detection of its expression and localization in hepatocellular carcinoma

ABSTRACTMIP is a candidate tumour suppressor protein found in hepatocellular carcinoma. It can interact with different proteins in different subcellular locations. However, antibodies against MIP for testing purposes are still not available and this has hindered the research on MIP protein. In this study, the full coding sequence of MIP protein was cloned into the prokaryotic expression vector pET-28a. The recombinant protein his-MIP was expressed by IPTG (isopropyl β-D-1-thiogalactopyranoside) induction and purified by nickel-affinity chromatography, then used to immunize New Zealand rabbits to obtain a polyclonal antibody against MIP. The sensitivity and specificity of the antibody were evaluated by enzyme-linked immunosorbent assay and western blot. We used the obtained antibody to detect endogenous MIP protein in hepatocellular carcinoma (HCC) cells and normal human hepatic cells L-O2 and found decreased MIP expression in HCC cells. The immunohistochemistry results further showed that the expression level of MIP protein in HCC tissues was lower than that in the corresponding adjacent non-cancerous tissues. The MIP protein was distributed mainly in the cytoplasm in HCC. In adjacent tissues, however, high expression of MIP was detected in the nucleus in addition to the cytoplasm. The prepared polyclonal antibody against MIP provides a useful tool for further research on the functions of MIP.