Human Liver Carcinoma Cell Line Research Articles

Genipin‐crosslinked microcarriers mediating hepatocellular aggregates formation and functionalities

In engineered regenerative medicine, various types of scaffolds have been customized to pursue the optimal environment for different types of therapeutic cells. In liver therapeutic research, hepatocytes require attachment to solid anchors for survival and proliferation before they could grow into cellular aggregates with enhanced functionalities. Among the various biomaterials scaffolds and vehicles, microspherical cell carriers are suited to these requirements. Individual spheres may provide two-dimensional (2D) cell-affinitive surfaces for cell adhesion and spreading; whereas multiple microcarriers may form three-dimensional (3D) matrices with inter-spherical space for cell expansion and multicellular aggregation. In this study, we culture human liver carcinoma cell line (HepG2) cells on genipin-crosslinked gelatin microspheres of two different sizes. Results suggest that both microcarriers support cell adhesion, proliferation, and spontaneous formation of hepatocellular aggregates, among which the spheres with bigger size (200-300 μm) seem more favorable than the smaller ones in terms of aggregate formation and liver specific functionalities. These findings suggest that the genipin-crosslinked microcarrier is a competent vehicle for liver cell delivery.

Antioxidant Activity of Hydrolysates and Peptide Fractions of Nemipterus japonicus and Exocoetus volitans Muscle

The focus of the study was to investigate the antioxidant activity of hydrolyzed muscle protein of Nemipterus japonicus and Exocoetus volitans. The trypsin protein hydrolysates of both fish showed maximum free radical scavenging potential and lipid peroxidation inhibition. Furthermore, it was purified by chromatographic methods followed by the lipid peroxidation inhibition; free radical scavenging assay was performed before and after purification. The purified peptide fractions of N. japonicus and E. volitans exhibited higher activity against polyunsaturated fatty acids (PUFA) peroxidation which was similar to natural antioxidants like α-tocopherol. Free radical scavenging potencies were measured by electron spin resonance technique (ESR). The purified peptide of E. volitans quenched free radicals (DPPH, hydroxyl, and superoxide) slightly more than N. japonicus. The amino acid composition of both fish protein hydrolysates showed variations in their ratio. The purified peptides were tested for cell cytotoxicity for Vero (kidney epithelial cells of the African Green Monkey) and Hep G2 (human hepatocellular liver carcinoma) cell lines. It was found that peptides did not show any cytotoxic effect for Vero cell lines and exerted a significant antiproliferative effect on Hep G2 cell lines.

Synthesis, characterization and in vitro antitumor activity of three organotin(IV) complexes with carbazole ligand

Three novel organotin(IV) complexes with 2-(9H-carbazol-9-yl) acetic acid ( HL), of the formulae {[ nBu 2SnOL] 2O} 2 ( 1), [ nBuSn(O)OL] 6 ( 2) and [ nBu 3SnOL] 6 ( 3) were prepared. All compounds were characterized by X-ray crystallography, confirming that complex ( 1) is tetranuclear one with ladder framework, complex ( 2) is a hexanuclear organotin(IV) complex with drum structure and complex ( 3) is a macrocycle with 24-membered stannoxane ring. Furthermore, all complexes were tested in vitro for their cytotoxic activity, using human hepatocellular carcinoma cell line (BEL-7402) and human hepatocellular liver carcinoma cell line (HepG2). Complex ( 1) displayed the best cytotoxicity and can be pointed out as a promising substrate to be subject of further investigations.

Synthesis and biological evaluation of some nitrogen containing steroidal heterocycles

epi-Androsterone 1 was converted into its hydrazone derivative through the reaction with hydrazine hydrate 80%. Hydrazonoandrostane derivative 2b reacted with hydrazonoyl halides in the presence of K 2CO 3 forming the corresponding hydrazopyridazinoandrostane derivatives 6a– d. The 3β-acetyl-17-hydrazonoandrostane derivative 2b reacted with a halogen reagent, benzoyl chloride, to form the non-cyclic 16-benzoylated hydrazone 9. On the other hand, compound 2b produced the corresponding pyridazinoandrostane derivatives 11 and 12 via its reaction with phenacyl bromide and chloroacetone respectively. Reaction of the hydrazono derivative 2b with benzaldehyde in the presence of acetic acid drops led to the formation of the benzylidenehydrazonoandrostane derivative 13. The product 14, phosphinom-ethylenehydrazonoandrostane was obtained by the reaction of the derivative 13 with trisdimethylaminophosphine in the presence of dry benzene. The reaction of compound 2b with phenyl isothiocyanate followed by boiling in chloroacetic acid or thioglycolic acid produced the pyrazoloandrostane derivatives 17 and 18 respectively. The biological activity of compounds 6a, 6d, 11, 12, and 15 was evaluated as inhibitor of growth in a human liver carcinoma cell line and doxorubicine was used for comparison. Compounds 15 and 12 showed a higher potency than the other tested compounds.

In vitro antitumor activity of N-glycosyl sulfonamides

A series of α-d-hex-2-enopyranosyl sulfonamides was evaluated for their antiproliferative activity against human hepatocellular liver carcinoma (HepG2) and human lung adenocarcinoma (A549) cell lines. The most potent compound (2,4,6-tri-O-acetyl-3-deoxy-α-d-erythro-hex-2-enopyranosyl ethanesulfonamide) showed antiproliferative properties in the micromolar range. The SARs of these sulfonamidoglycoside which includes the influence of carbohydrate rings and sulfonamide class are described.

Synthesis, characterization, and biological activity of a Cu(I) complex with 2-(9H-carbazol-9-yl) acetic acid

A new dicopper(I) complex with 2-(9H-carbazol-9-yl) acetic acid (HL) of the formula [Cu2(dppm)2L(NO3)(CH3OH)] [dppm = bis(diphenylphosphino)methane] was prepared. The complex was structurally characterized by IR, 1H NMR spectra, and elemental analysis. Single crystal X-ray crystallography revealed that this complex is monoclinic, space group P21/c, with a = 13.6552(17) Å, b = 23.123(2) Å, c = 19.257(2) Å, α = γ = 90.00°, β = 106.860(2)°, V = 5818.8(11) Å3, Z = 4, D Calcd = 1.386 mg m−3, F(000) = 2512, goodness-of-fit = 1.015. The complex was also tested in vitro for its cytotoxic activity using human hepatocellular carcinoma cell line (BEL-7402) and human hepatocellular liver carcinoma cell line (Hep-G2); 5-Fluorouracil was used as a positive control substance. The results indicated that the complex exhibited good cytotoxic activity against both human tumor cell lines.

Delivery of Anticancer Drugs Using Polymeric Micelles Stabilized by Hydrogen‐Bonding Urea Groups

Polymeric micelles comprising a hydrogen-bonding core were formed from block copolymers with pendant urea groups and explored as drug delivery vehicles. The amphiphilic block copolymers were synthesized by organocatalytic ring opening polymerization (ROP) of urea-functionalized cyclic carbonates from a poly(ethylene glycol) macroinitiator. The urea functionality was incorporated because its ability to increase the hydrophobic core's affinity toward polar organic compounds through intermolecular hydrogen bonding. Doxorubicin (DOX), a lipophilic anticancer drug with hydrogen-bonding functionalities, was systematically incorporated into the micelle's hydrophobic interior via hydrogen bonding to the functionalized monomers. Micelles employing urea groups were found to more efficiently interact with DOX thus allowing increased drug loading capacity while maintaining a desirable micellular size. More importantly, while DOX-loaded micelles were shown to kill HepG2 human liver carcinoma cell lines efficiently, all of the polymers were non-cytotoxic.

Disassemblable micelles based on reduction-degradable amphiphilic graft copolymers for intracellular delivery of doxorubicin

Disassemblable micelles for intracellular delivery of doxorubicin were developed based on a reduction-degradable amphiphilic polyamide amine- g-polyethylene glycol graft copolymer containing disulfide linkages throughout the main chain. The micelles are spherical of less than 50 nm in diameter, and can load doxorubicin in the core with drug loading content up to 20%. The micelles are stable in normal physiological condition, and quickly disassemble in reductive condition due to the cleavage of the disulfide linkages. The drug release of the micelles in normal condition is less than 25% within 24 h, whereas in the presence of reductive agent, DTT, the micelles can quickly release the entire loaded drug within 10 h. CLSM observation showed that the micelles can effectively deliver the drug cargo into nuclei after internalized through endocytosis. Cytotoxicity of the drug-loaded disassemblable micelles was demonstrated using human cervical cancer cell line (HeLa) and human liver carcinoma cell line (HepG2).

Dynamic manipulation and patterning of microparticles and cells by using TiOPc-based optoelectronic dielectrophoresis

We develop light-driven optoelectronic tweezers based on the organic photoconductive material titanium oxide phthalocyanine. These tweezers function based on negative dielectrophoresis (nDEP). The dynamic manipulation of a single microparticle and cell patterning are demonstrated by using this light-driven optoelectronic DEP chip. The adaptive light patterns that drive the optoelectronic DEP onchip are designed by using Flash software to approach appropriate dynamic manipulation. This is also the first reported demonstration, to the best of our knowledge, for successfully patterning such delicate cells from human hepatocellular liver carcinoma cell line HepG2 by using any optoelectronic tweezers.

Polycyclic aromatic hydrocarbons (PAHs) in different indoor dusts and their potential cytotoxicity based on two human cell lines

In this study, 55 air-conditioner filter dust samples from six different workplaces including commercial office, secondary school, shopping mall, hospital, electronic factory and manufacturing plant in Hong Kong were collected for analyses of PAH concentration and cytotoxicity. Chemical analyses showed that the total PAHs ranged from 1.17 to 25.5 μg/g, with the dust samples from manufacturing plant having the highest concentration. MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrasodium bromide) assay was performed to evaluate the cytotoxicity of organic dust extracts using human hepatocellular liver carcinoma cell line (HepG2) and human skin keratinocyte cell line (KERTr). Each organic dust extract showed marked dose-related response. Dust samples from the manufacturing plant showed the highest cytotoxicity. Curve estimation indicated that power model was fit for explaining the relationship between the total PAH concentration and lethal concentration 50 (LC 50). In addition, a significant negative correlation was observed between the total PAH concentration and LC 50 both on HepG2 ( r = −0.65, p < 0.01) and KERTr ( r = −0.63, p < 0.01) cell lines. Source analyses demonstrated that the PAHs in dust were derived from pyrogenic origins.

Nutrient regulation of bacterial growth and production of anti-tumor metabolites in Stigmatella WXNXJ-B fermentation

The metabolites produced by Stigmatella WXNXJ-B inhibited the growth of tumor cells. The aims of this research were to evaluate the inhibition potency to different tumor cell lines and to study the effects of ammonium, phosphate and iron salts on bacterial growth and production of bioactive metabolites in Stigmatella WXNXJ-B fermentation. The results showed that the chloroform extract (CE-ME) showed the strongest growth inhibition bioactivity on mouse melanoma cell line (B16), murine colon carcinoma cell line (CT-26), human liver carcinoma cell line (HepG2) and human breast cancer cell line (MDA-MB231) in vitro and the IC50 values were 9.94, 7.33, 11.34 and 11.66 μg ml−1 respectively. The IC50 value was above 700 μg ml−1 on normal mouse spleen cells. Morphology happened changes in B16 cells treated with CE-ME. The anti-tumor metabolites were mainly produced during the stationary phase of the bacterial growth. Cell growth was stimulated at the phosphate concentration below 5 mM, but it was inhibited partly with 10 mM phosphate. The production of bioactive substances was inhibited by the phosphate. Ammonium increased the cell growth by 250% at 5 mM addition. The inhibition rate to B16 cells was increased to 89% at the concentration of 40 mM ammonium. The bacteria showed the best growth with 4 mM iron. Iron had little effect on the production at 2 mM, but bigger inhibition effect at higher iron concentration.

PiggyBac Transposon-mediated Long-term Gene Expression in Mice

Transposons are promising systems for somatic gene integration because they can not only integrate exogenous genes efficiently, but also be delivered to a variety of organs using a range of transfection methods. piggyBac (PB) transposon has a high transposability in mammalian cells in vitro, and has been used for genetic and preclinical studies. However, the transposability of PB in mammalian somatic cells in vivo has not been demonstrated yet. Here, we demonstrated PB-mediated sustained gene expression in adult mice. We constructed PB-based plasmid DNA (pDNA) containing reporter [firefly and Gaussia luciferase (Gluc)] genes. Mice were transfected by injection of these pDNAs using a hydrodynamics-based procedure, and the conditions for high-level sustained gene expression were examined. Consequently, gene expressions were sustained over 2 months. Our results suggest that PB is useful for organ-selective somatic integration and sustained gene expression in mammals, and will contribute to basic genetic studies and gene therapies.

α-Bisabolol induces dose- and time-dependent apoptosis in HepG2 cells via a Fas- and mitochondrial-related pathway, involves p53 and NFκB

In this study, the apoptotic effect of α-bisabolol, a sesquiterpene, against human liver carcinoma cell line HepG2 was investigated. MTT assay showed α-bisabolol could effectively induce cytotoxicity in several human cancer cell lines (PC-3, Hela, ECA-109 and HepG2). The results of nuclei morphology examination, DNA fragmentation detection, flow cytometry analysis and cleavage of poly(ADP-ribose) polymerase and caspases indicated α-bisabolol might induce dose- and time-dependent apoptosis in HepG2 cells. Western blot data also showed a cascade activation of caspases-8,-9,-3 and promoted expression of Fas, implying caspase-8 might function as an upstream regulator, and the Fas-related pathway might be involved in this process. Preparation of mitochondrial/cytosol fraction followed with immunoblot analysis showed the release of chromosome c from mitochondria, down-regulated expression of Bcl-2 and translocation of Bax, Bak and Bid, suggesting the mitochondrial-related pathway might be involved in α-bisabolol-induced apoptosis either. Detection of accumulation of nuclear wild-type p53 and up-regulated expression of NFκB indicated these two key regulator with transcriptional decision-making function in various signaling pathways might also play a role in α-bisabolol-induced apoptosis in HepG2 cells.

Synthesis and antiproliferative activity of indolizine derivatives incorporating a cyclopropylcarbonyl group against Hep-G2 cancer cell line

Indolizine and annulated indolizine derivatives incorporating a cyclopropylcarbonyl group were synthesized in a one pot procedure by the tanden reactions of [3 + 2] cycloaddition of the corresponding N-ylide with electron deficient alkene. Seventeen indolizine derivatives were reported for the first time. All the compounds were examined for their antiproliferative activity against the human hepatocellular liver carcinoma ( Hep-G2) cell line by MTT method. Among the compounds tested, 5a, 5d, 5g and 5j showed the most favorable activities with IC 50 values of 0.39, 0.48, 0.29 and 0.20 μg/mL. Especially, compound 5j displayed potent antiproliferative activities with IC 50 value of 0.20 μg/mL, and showed significant EGFR kinase inhibitory activity with IC 50 value of 0.085 μM. Docking simulations of 5j were carried out to illustrate the binding mode of the molecular into the EGFR active site.

Phylogenetic diversity of culturable fungi associated with two marine sponges: Haliclona simulans and Gelliodes carnosa, collected from the Hainan Island coastal waters of the South China Sea

The diversity and biological activities of fungi associated with the two sponges, Haliclona simulans and Gelliodes carnosa, were investigated using a culture-dependent method followed by analysis of the fungal rDNA-ITS sequences. The two sponges were collected from the coastal waters of Lingshui Bay of Hainan Island in the South China Sea. A total of 37 independent fungal isolates corresponding to 30 different species were obtained from the two sponges. Nearly two thirds of the strains (n = 24, 64.9%) had close affiliations (identity (ID) or similarity ≥ 98%) with their best matches in GenBank. Another one third of the isolates (n = 13, 35.1%) were distantly related to their closest relatives (ID < 98%), implying that these species are possibly different from those previously reported. The two sponges possessed similar fungal diversities. Haliclona simulans harbored a mainly different fungal consortium as compared with that of the same sponge species collected from Irish coastal waters, suggesting that the fungal diversity associated with the sponges is more dependent on the surrounding environment than on the sponge species. Biological activities of the fungal culture extracts were tested against the human tumor cell lines, mainly, a human lung carcinoma cell line (A-549), a human liver carcinoma cell line (Bel-7402), and a human colon carcinoma cell line (HCT-8), and against the Gram positive bacterium Bacillus subtilis. A relatively high proportion of positive results were obtained in this study, demonstrating that fungi isolated from sponges could be a rich source of new biologically active natural products.

Cell cycle and size sorting of mammalian cells using a microfluidic device

Microfluidic devices can sort viable mammalian cells by size. In this study, we investigated size-based sorting of cells using flow splitting microfluidic devices based on hydrodynamic filtration for noninvasive cell cycle synchronization. Two different types of mammalian cell lines, HepG2 (human hepatocellular liver carcinoma cell line) and NIH/3T3 (mouse embryonic fibroblast cell line) were sorted by microfluidic device and its DNA contents were analyzed. Our results showed that a microfluidic device can synchronize the cell cycle after size separation. The damage-free separation of living cells in different phases of the cell cycle represents a potentially promising technology for the investigation of gene transfection and gene expression.

Combined effect of high mobile group box-1 protein and lipopolysaccharide on release of cytokines from human HepG2 cells

To study the effects of high mobile group box-1 protein (HMGB1) and lipopolysaccharide (LPS) singly or in combination on release of cytokines from human liver carcinoma cell line (HepG2). HepG2 cells were cultured, and purified HMGB1 protein was prepared by chromatography on Ni(2+)-NTA Sepharose column under natural conditions with recombinant expression plasmid pET14b-HMGB1. Different concentrations of HMGB1 (0, 0.01, 0.1, 1, 10 mg/L) and LPS (0, 0.1, 1, 10, 100 mg/L) were added into the cultured cells for 24 hours, respectively. Then the supernatant were collected to detect the levels of granulocyte/macrophage colony stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, IL-8, IL-10, and IL-12 by using LiquiChip system. Lastly, HepG2 cells were co-stimulated with 10 mg/L LPS and 1 mg/L HMGB1 for 24 hours. The supernatant were collected to determine the levels of above ten of cytokines . The expression and release of IL-6 and IL-8 increased from HepG2 cells after being stimulated by LPS in a dose-dependent manner, but there were no changes in other eight kinds of cytokines (P<0.05 or P<0.01). Low concentration of HMGB1 could up-regulate the expression of IL-6 and IL-8 in HepG2 cells (both P<0.01). But the extent of induction decreased with higher concentration of HMGB1. Similar to LPS, there was no effect of HMGB1 on the expression of other eight kinds of cytokines from cultured HepG2 cells. Furthermore, high concentration of HMGB1 could obviously inhibit the upregulation of IL-6 and IL-8 by high concentration of LPS when the HepG2 cells were co-stimulated with LPS and HMGB1 (both P<0.01). HepG2 cells could only express and release a few kinds of cytokines when the cells were stimulated with pro-inflammatory agents, such as LPS or HMGB1. Two kinds of cytokines, IL-6 and IL-8 could be up-regulated by LPS and low concentration of HMGB1, and HMGB1 acted as an inhibitor of LPS to down-regulate the expression and release of IL-6 and IL-8 from HepG2 cells.

Synthesis and cytotoxic analysis of some disodium 3β,6β-dihydroxysterol disulfates

Disodium 3β,6β-dihydroxy-5α-cholestane disulfate ( 1) was synthesized in 4 steps with a high overall yield from cholesterol. First, cholesterol ( 4a) was converted to cholest-4-en-3,6-dione ( 5a) via oxidation with pyridinium chlorochromate (PCC) and then 5a was reduced by NaBH 4 in the presence of NiCl 2 to produce cholest-3β,6β-diol ( 6a). The reaction of 6a with the triethylamine-sulfur trioxide complex generated diammonium 3β,6β-dihydroxy-5α-cholestane disulfate ( 7a) and the treatment of 7a by cation exchange resin 732 (sodium form)(Na +) yielded the target steroid 1. Disodium 24-ethyl-3β,6β-dihydroxycholest-22-ene disulfate ( 2) and disodium 24-ethyl-3β,6β-dihydroxycholestane disulfate ( 3) were synthesized using a similar method. The cytotoxicity of these compounds against Sk-Hep-1 (human liver carcinoma cell line), H-292 (human lung carcinoma cell line), PC-3 (human prostate carcinoma cell line) and Hey-1B (human ovarian carcinoma cell line) cells was investigated. Our results indicate that presence of a cholesterol-type side chain at position 17 is necessary for their biological activity.

Potent Antitumor Effect of Interleukin-24 Gene in the Survivin Promoter and Retinoblastoma Double-Regulated Oncolytic Adenovirus

Interleukin (IL)-24 is an excellent therapeutic gene for cancer therapy. In this work, IL-24 was inserted into Ad.sp-E1A(Delta24), an oncolytic adenovirus with a 24-bp deletion in the E1A gene, which was driven by the survivin promoter to form Ad.sp-E1A(Delta24)-IL-24. Ad.sp-E1A(Delta24)-IL-24 has an excellent antitumor effect in vitro for human nasopharyngeal, liver, lung, and cervical carcinoma cell lines but does no or little damage to normal cell lines L-02 and WI38. Furthermore, it achieved nearly complete inhibition (although not elimination) of NCI-H460 lung carcinoma growth in nude mice. The antitumor efficacy of Ad.sp-E1A(Delta24)-IL-24 on NCI-H460 cells was clearly mediated by apoptosis, because it induced caspase-3 and poly(ADP-ribose) polymerase cleavage. This is the first report of Ad.sp-E1A(Delta24)-IL-24 with such an excellent, broad, and specific antitumor effect in vitro and nearly complete inhibition of lung tumor growth in vivo.

Quantitative proteomic analysis of a paired human liver healthy versus carcinoma cell lines with the same genetic background to identify potential hepatocellular carcinoma markers

To comprehensively measure global changes in protein expression associated with human hepatocellular carcinoma (HCC), comparative proteomic analysis of two cell lines derived from the healthy and carcinoma tissue of a same donor respectively was conducted using quantitative amino acid-coded mass tagging /stable isotope labeling with amino acids in cell culture-based LC-MS/MS approach. Among a total of 501 proteins precisely quantified, the expressions of 128 proteins were significantly altered including 70 proteins up-regulated and 58 down-regulated in HCC cells. According to their previously characterized functions, the differentially expressed proteins were found associated with nine functional categories including glycolysis, stress response, cell communication, cell cycle, apoptosis/death, etc. For example, multiple enzymes involving glycolysis pathway were found differentially regulated in HCC cells, illustrating the critical participation of glycolysis in the HCC transformation. The accuracy of certain differentially expressed proteins identified through the amino acid-coded mass tagging-based quantification was validated in the paired cell lines, and later their pathological correlations were examined in multiple clinical pairs of normal versus tumor tissues from HCC specimen by using a variety of biological approaches including Western blotting and in situ immunoassays. These consistencies suggested that multiple proteins such as HSP27, annexin V, glyceraldehyde-3-phosphate dehydrogenase, nucleolin and elongation factor Tu could be the biomarkers candidates for diagnosis of HCC.