Live cell imaging of highly activated natural killer cells against human hepatocellular carcinoma in vivo

Background aimsTracking administered natural killer (NK) cells in vivo is critical for developing an effective NK cell-based immunotherapy against human hepatocellular carcinoma (HCC). Here the authors established a new molecular imaging using ex vivo-activated NK cells and investigated real-time biodistribution of administered NK cells during HCC progression.MethodsEx vivo-expanded NK cells from healthy donors were labeled with a near-infrared lipophilic cytoplasmic dye, and their proliferation, surface receptor expression and cytotoxicity activity were evaluated. Human HCC HepG2 cells were implanted into the livers of NOD.Cg-Prkdc^scid IL2rg^tm1Wjl/SzJ (NSG) mice. The authors administered 1,1’-dioctadecyltetramethyl indotricarbocyanine iodide (DiR)-labeled NK cells intravenously to non-tumor-bearing and intrahepatic HCC tumor-bearing NSG mice. Fluorescent imaging was performed using a fluorescence-labeled organism bioimaging instrument. Single cell suspensions from the resected organs were analyzed using flow cytometry.ResultsThe fluorescent DiR dye was nontoxic and did not affect the proliferation or surface receptor expression levels of the NK cells, even at high doses. The administered DiR-labeled NK cells immediately migrated to the lungs of the non-tumor-bearing NSG mice, with increased NK cell signals evident in the liver and spleen after 4 h. NK cells migrated to the intrahepatic tumor-bearing livers of both early- and late-stage HCC mice within 1 h of injection. In early-stage intrahepatic tumor-bearing mice, the fluorescence signal increased in the liver until 48 h post-injection and decreased 7 days after NK injection. In late-stage HCC, the NK cell fluorescence signal was the highest in the liver for 7 days after NK injection and persisted for 14 days. The purity of long-term persistent CD45⁺CD56⁺CD3⁻ NK cells was highest in early- and late-stage HepG2-bearing liver compared with normal liver 2 weeks after NK injection, whereas highest purity was still observed in the lungs of non-tumor-bearing mice. In addition, Ki-67 expression was detected in migrated human NK cells in the liver and lung up to 72 h after administration. With HepG2 tumor progression, NK cells reduced the expression of NKp30 and NKG2D.ConclusionsAdministered NK cells were successfully tracked in vivo by labeling the NK cells with near-infrared DiR dye. Highly expanded, activated NK cells migrated rapidly to the tumor-bearing liver, where they persisted for 14 days after administration, with high purity of CD45⁺CD56⁺CD3⁻ NK cells. Liver biodistribution and persistence of administered NK cells showed significantly different accumulation patterns during HCC progression.     

Spectroscopic investigation and in vitro cytotoxic activity toward HepG2 cells of a copper compound complexed with human serum albumin

The human serum albumin (HSA) interaction of a mixed‐ligand copper compound (1) with an imidazole and taurine Schiff base derived from salicylaldehyde and taurine was investigated using fluorescence spectroscopy, UV–vis spectroscopy, time‐resolved fluorescence spectroscopy, circular dichroism (CD) spectroscopy, Fourier transform infrared (FT‐IR) spectroscopy and a molecular docking technique. The results of fluorescence and time‐resolved fluorescence spectroscopy indicated that 1 can effectively quench the HSA fluorescence by a static mechanism. Binding constants (K) and the number of binding sites (n ≈ 1) were calculated using modified Stern–Volmer equations. The thermodynamic parameters were calculated. UV–vis, CD and FT‐IR spectroscopy measurements confirm the alterations in the HSA secondary structure induced by 1. The site marker competitive experiment confirms that 1 is located in subdomain IB of HSA. The combination of molecular docking results and fluorescence experimental results reveal that hydrophobic interaction and hydrogen bonds are the predominant intermolecular forces stabilizing the 1–HSA complex. The 1–HSA complex increases approximately three times its cytotoxicity in cancer cells but has no effect on normal cells in vitro. Compared with unbound 1, the 1–HSA complex promotes HepG2 cells apoptosis and also has a stronger capacity for cell cycle arrest at the S phase of HepG2 cells.     

Synthesis of functionalized amino acid derivatives as new pharmacophores for designing anticancer agents

A new series of functionalized amino acid derivatives N-substituted 1-N-(tert-butoxycarbonyl)-2,2-dimethyl-4-phenyl-5-oxazolidine carboxamide (1-17) and 1-N-substituted-3-amino-2-hydroxy-3-phenylpropane-1-carboxamide (18-34) were synthesized and evaluated for their in vitro cytotoxicity against human cancer cell lines. Compound 6 has shown interesting cytotoxicity (IC₅₀ = 5.67 μm) in ovarian cancer, while compound 10 exhibited promising cytotoxicity in ovarian (IC₅₀ = 6.1 μm) and oral (IC₅₀ = 4.17 μm) cancers. These compounds could be of use in designing new anti-cancer agents.     

Cobalt (II) complex with novel unsymmetrical tetradentate Schiff base (ON) ligand: in vitro cytotoxicity studies of complex,interaction with DNA/protein,molecular docking studies,and antibacterial activity

[C₂₀H₁₇N₃O₂] and cobalt (II) complex [Co(L²)(MeOH)₂].ClO₄, (L² = 4-((E)-1-((2-(((E)-pyridin-2-ylmethylene) amino) phenyl) imino) ethyl) benzene-1, 3-diol) novel Schiff base has been synthesiszed and chracterized by Fourier transform infrared, UV–vis, ¹H-NMR spectroscopy, and elemental analysis techniques. The interaction of Co(II) complex with DNA and BSA was investigated by electronic absorption spectroscopy, fluorescence spectroscopy, circular dichroism, and thermal denaturation studies. Our experiments indicate that this complex could strongly bind to CT-DNA via minor groove mechanism. In addition, fluorescence spectrometry of BSA with the complex showed that the fluorescence quenching mechanism of BSA was of static type. The complex exhibited significant in vitro cytotoxicity against three human cancer cell lines (JURKAT, SKOV3, and U87). The molecular docking experiment effectively proved the binding of complex to DNA and BSA. Finally, antibacterial assay over gram-positive and gram-negative pathogenic bacterial strains was studied.     

An improved resazurin-based cytotoxicity assay for hepatic cells

A simple resazurin-based cytotoxicity assay is presented for screening of cytotoxicity in hepatocytes and liver cell lines. Human hepatoma (HepG2) cells in 96-well culture plates were exposed to known toxic (cisplatin, 5-fluorouracil, ethionine, flufenamic acid, and diflunisal) and control (transplatin, 5-chlorouracil, methionine, and acetylsalicylic acid) compounds for 1–3 days, and resazurin (5 μmol/L) was added. A conventional short-term (1 h) assay was first performed, where cytotoxicity is indicated by decreased reduction of resazurin to its fluorescent product resorufin. Our improved assay consists of additionally measuring fluorescence 2–4 days later, when cytotoxicity is indicated by a striking increase in the concentration of resorufin, resulting from two distinct processes. First, viable liver-derived cells slowly convert resorufin to nonfluorescent metabolites. Fluorescence of control cell wells decreased to background during a 2- to 4-day exposure to resazurin. This metabolism of resorufin was largely blocked by dicumarol and to lesser extents by disulfiram and SKF525a. Second, dead or dying cells slowly convert resazurin to resorufin but do not further metabolize resorufin; thus this fluorescent metabolite accumulates to high levels in wells with dead cells by 2 to 4 days. A similar increase in fluorescence associated with cytotoxicity was observed in primary cultures of rat hepatocytes using the long-term resazurin-based assay. In addition to an improved signal relative to the short-term assay, the inversion of the fluorescent signal from high = alive short-term to high = dead long-term allows determination of two independent cytotoxicity endpoints after addition of one innocuous vital dye. This revised version was published online in July 2006 with corrections to the Cover Date.     

A potent and specific immunotoxin for tumor cells expressing disialoganglioside GD2

Summary Monoclonal antibody 14G2a (anti-GD₂) reacts with cell lines and tumor tissues of neuroectodermal origin that express disialoganglioside GD₂. mAb 14G2a was coupled to the ribosome-inactivating plant toxin gelonin with the heterobifunctional cross-linking reagentN-succinimidyl-3(2-pyridyldithio)propionate. The activity of the immunotoxin was assessed by a cell-free translation assay that confirmed the presence of active gelonin coupled to 14G2a. Data from an enzyme-linked immunosorbent assay demonstrated the specificity and immunoreactivity of the 14G2a-gelonin immunotoxin, which was identical to that of native 14G2a. Assays for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) revealed that these functional properties of the native 14G2a antibody were also preserved in the 14G2a-gelonin immunotoxin. The gelonin-14G2a immunotoxin was directly cytotoxic to human melanoma (A375-M and AAB-527) cells and was 1000-fold more active than native gelonin in inhibiting the growth of human melanoma cells in vitro. The augmentation of tumor cell killing of 14G2a-gelonin immunotoxin was examined with several lysosomotropic compounds. Chloroquine and monensin, when combined with 14G2a-gelonin immunotoxin, augmented its cytotoxicity more than 10-fold. Biological response modifiers such as tumor necrosis factor and interferon and chemotherapeutic agents such as cisplatinum andN,N-bis(2-chloroethyl)-N-nitrosourea (carmustine) augmented the cytotoxicity of 14G2a-gelonin 4- to 5-fold. The results of these studies suggest that 14G2a-gelonin may operate directly by both cytotoxic efforts and indirectly by mediating both ADCC and CDC activity against tumor cells; thus it may prove useful in the future for therapy of human neuroectodermal tumors.Research conducted, in part, by the Clayton Foundation for Research     

Vanadium compounds discriminate hepatoma and normal hepatic cells by differential regulation of reactive oxygen species

Our previous study indicated that vanadium compounds can block cell cycle progression at the G1/S phase in human hepatoma HepG2 cells via a highly activated extracellular signal-regulated protein kinase (ERK) signal. To explore their differential action on normal cells, we investigated the response of an immortalized hepatic cell line, L02 cells. The results demonstrated that a higher concentration of vanadium compounds was needed to inhibit L02 proliferation, which was associated with S and G2/M cell cycle arrest. In addition, in contrast to insignificant reactive oxygen species (ROS) generation in HepG2 cells, all of the vanadium compounds resulted significant increases in both O ₂ ^·? and H₂O₂ levels in L02 cells. At the same time, ERK and c-Jun N-terminal kinase (JNK) as well as cell division control protein 2 homolog (Cdc2) were found to be highly phosphorylated, which could be counteracted with the antioxidant N-acetylcysteine (NAC). The current study also demonstrated that both the ERK and the JNK pathways contributed to the cell cycle arrest induced by vanadium compounds in L02 cells. More importantly, it was found that although NAC can ameliorate the cytotoxicity of vanadium compounds in L02 cells, it did not decrease their cytotoxicity in HepG2 cells. It thus shed light on the potential therapeutic applications of vanadium compounds with antioxidants as synergistic agents to reduce their toxicities in human normal cells without affecting their antitumor activities in cancer cells.     

Transcriptome analysis of signaling pathways targeted by Ellagic acid in hepatocellular carcinoma cells

BackgroundEllagic acid (EA) possesses prominent inhibitory activities against various cancers, including hepatocellular carcinoma (HCC). Our recent study demonstrated EA's activities in reducing HCC cell proliferation and tumor formation. However, the mechanisms of EA to exert its anticancer activities and its primary targets in cancer cells have not been systematically explored.MethodsCell proliferation assay and flow cytometric analysis were used to examine the effects of EA treatment on viability and apoptosis, respectively, of HepG2 cells. RNA-seq studies and associated pathway analyses by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were employed to determine EA's primary targets. Differentially expressed genes (DEG) in EA-treated HepG2 cells were verified by RT-qPCR and Western blot. Integrative analyses of the RNA-seq dataset with a TCGA dataset derived from HCC patients were conducted to verify EA-targeted genes and signaling pathways. Interaction network analysis of the DEGs, shRNA-mediated knockdown, cell viability assay, and colony formation assay were used to validate EA's primary targets.ResultsEA reduced cell viability, caused DNA damage, and induced cell cycle arrest at G1 phase of HepG2 cells. We identified 5765 DEGs encoding proteins with over 2.0-fold changes in EA-treated HepG2 cells by DESeq2. These DEGs showed significant enrichment in the pathways regulating DNA replication and cell cycle progression. As primary targets, p21 was significantly upregulated, while MCM2–7 were uniformly downregulated in response to EA treatment. Consistently, p21 knockdown desensitized liver cells to EA in cell viability and colony formation assays.ConclusionEA induced G1 phase arrest and promoted apoptosis of HCC cells through activating the p21 gene and downregulating the MCM2–7 genes, respectively.General significanceThe discoveries in this study provide helpful insights into developing novel strategies in the therapeutic treatment of HCC patients.     

Anticancer mechanisms of temporin-1CEa,an amphipathic α-helical antimicrobial peptide,in Bcap-37 human breast cancer cells

AimsTemporin-1CEa, a 17-residue antimicrobial peptide, is known to exert broad-spectrum anticancer activity that acts preferentially on cancer cells instead of normal cells. However, the mechanism of cancer cell death induced by temporin-1CEa is weakly understood.Main methodsHere, we investigated the cytotoxic and membrane-disrupting effects of temporin-1CEa on human breast cancer cell line Bcap-37, using MTT assay, electronic microscope observation, fluorescence imaging and flow cytometry analysis.Key findingsThe MTT assay indicated that one-hour temporin-1CEa treatment led to rapid cell death in either caspase-dependent or -independent manner. The electronic microscope observation suggested that temporin-1CEa exposure resulted in profound morphological changes in Bcap-37 cells. The fluorescence imaging and flow cytometry analysis demonstrated that temporin-1CEa exhibited membrane-disrupting property characterized by induction of cell-surface phosphatidylserine exposure, elevation of plasma membrane permeability, and rapid transmembrane potential depolarization. Moreover, temporin-1CEa might also induce rapid cell death through mitochondria-involved mechanisms, including rapid intracellular Ca^2 + leakage, collapse of mitochondrial membrane potential (Δφm) and over-generation of reactive oxygen species (ROS).SignificanceIn summary, the present study indicates that temporin-1CEa triggers a rapid cytotoxicity in Bcap-37 cells through membrane-destruction and intracellular mechanisms involving mitochondria. These intracellular mechanisms and direct membrane-destruction effect were evaluated helping to understand the detail action of antimicrobial peptides in mammalian cancer cells.     

Anti-hyperglycemic effects of Eryngium billardierei F. Delaroche extract on insulin-resistance HepG2 cells in vitro

Background

Insulin resistance as a major problem is associated with type 2 diabetes mellitus. This study investigated the effect of Eryngium billardierei on insulin-resistance induced HepG2 cells.

Methods and results

MTT method was used to evaluate the viability of HepG2 cells treated with various doses of E. billardierei extract. An insulin-resistance model was established in HepG2 cells. Next, MTT assay and Acridine orange staining were performed to investigate the viability of cells in the vicinity of different concentrations of insulin, pioglitazone, and E. billardierei extract in an insulin-resistance media. The glucose uptake test was performed to select the optimal insulin concentration. Expression levels of IR, G6Pase, and PEPCK genes were assessed by real-time RT-PCR. According to obtained data, E. billardierei at concentrations of 0.5 and 1 mg/mL show no toxicity on cells. Furthermore, based on MTT assay and glucose uptake test 10^?5 mol/L insulin was chosen as the model group to induce insulin-resistance in HepG2 cells for gene expression analysis. Finally, 1 mg/mL E. billardierei not only induced no cytotoxicity but also showed an increase in the expression of IR as well as a reduction in G6Pase and PEPCK level compared to the control and model groups.

Conclusions

The obtained data indicated that 1 mg/mL E. billardierei might have an anti-insulin resistance effect on insulin-resistance HepG2 cells in vitro and could be a promising candidate with anti-hyperglycemic properties for diabetes treatments.

Graphical abstract

     

DNA lesions and cytogenetic changes induced by N-nitrosomorpholine in HepG2, V79 and VH10 cells: the protective effects of Vitamins A, C and E

Introduction: N-Nitrosomorpholine (NMOR), present in the workplace of tyre chemical factories, is a known hepatocarcinogen. This compound belongs to the group of N-nitrosamines, which are indirect-acting and require metabolic activation. However, the mechanism of its carcinogenic effect is not completely clear. Aims: The objective of this study was (i) to compare the DNA-damaging and clastogenic effects of NMOR in three cell lines (HepG2, V79 and VH10) with different levels of metabolizing enzymes and (ii) to determine the protective effects of Vitamins A, C and E against deleterious effects of NMOR. Methods: The exponentially growing cells were pre-treated with Vitamins A, C and E and treated with NMOR. Genotoxic effects of NMOR were evaluated by single-cell gel electrophoresis (SCGE, comet assay), while the chromosomal aberration assay was used for the study of clastogenic effects. Key results: NMOR-induced a significant dose-dependent increase of DNA damage as analyzed by SCGE, but the extent of DNA migration in the electric field was unequal in the different cell lines. Although the results obtained by SCGE confirmed the genotoxicity of NMOR in all cell lines studied, the number of chromosomal aberrations was significantly increased only in HepG2 and V79 cells, while no changes were observed in VH10 cells. In HepG2 cells pre-treated with Vitamins A, C and E we found a significant decrease of the percentage of tail DNA induced by NMOR. The reduction of the clastogenic effects of NMOR was observed only after pretreatment with Vitamins A and E; Vitamin C did not alter the frequency of NMOR-induced chromosomal aberrations under the experimental conditions of this study. Conclusions: The fat-soluble Vitamins A and E, which are dietary constituents, reduce the harmful effects of N-nitrosomorpholine in human hepatoma cells HepG2, which are endowed with the maximal capacity for metabolic activation of several drugs.     

Taiwanin A induced cell cycle arrest and p53-dependent apoptosis in human hepatocellular carcinoma HepG2 cells

Taiwanin A, a lignan isolated from Taiwania cryptomerioides Hayata, has previously been reported to have cytotoxicity against human tumor cells, but the mechanisms are unclear. In this study, we examined the molecular mechanism of cell death of human hepatocellular carcinoma HepG2 cells induced by Taiwanin A. Taiwanin A has been found to induce cell cycle arrest at G2/M phase as well as caspase-3-dependent apoptosis within 24 h. We performed both in vitro turbidity assay and immunofluorescence staining of tubulin to show that Taiwanin A can inhibit microtubule assembly. Moreover, the tumor suppressor protein p53 in HepG2 cells was activated by Taiwanin A within 12 h. Inhibition of p53 by either pifithrin-alpha or by short hairpin RNA which blocks p53 expression attenuates Taiwanin A cytotoxicity. Our results demonstrate that Taiwanin A can act as a new class of microtubule damaging agent, arresting cell cycle progression at mitotic phase and inducing apoptosis through the activation of p53.     

ZL11n is a novel nitric oxide-releasing derivative of farnesylthiosalicylic acid that induces apoptosis in human hepatoma HepG2 cells via MAPK/mitochondrial pathways

ZL11n is a novel furoxan-based nitric oxide (NO)-releasing derivative of farnesylthiosalicylic acid. In this study, we examined the anticancer effects and the potential mechanism of action of ZL11n in vitro and in vivo. It was found that ZL11n exhibited a favorable, selective cytotoxic effect in the HepG2 cell line. The yield of NO in the ZL11n treated HepG2 cells was much higher than in the control group and the normal human liver L-02 cells. Furthermore, the NO concentration was correlated to the degree of cytotoxicity observed. The ZL11n-induced apoptosis was assessed by Annexin V-FITC/propidium iodide flow cytometry assay. ZL11n triggered the mitochondrial/caspase apoptotic pathway by decreasing mitochondrial membrane potential, cytochrome c release from mitochondrial, and reducing the Bcl-2-to-Bax ratio, in addition to activating the caspase cascade. Simultaneously, we found that ZL11n treatment led to an increase in JNK and ERK1/2 phosphorylation. Furthermore, treatment with SP600125 (a JNK inhibitor) and PD98059 (an ERK1/2 inhibitor) prior to ZL11n treatment was found to significantly reverse ZL11n-induced apoptosis. The in vivo findings also revealed that ZL11n significantly reduced tumor volume and weight in the H₂₂ solid tumor mouse model examined. In short, our findings suggest that ZL11n induced apoptosis through the coordination of the mitochondrial apoptotic pathway (activated by NO) and MAPKs signaling pathway (triggered by JNK or ERK).     

Characteristics of the killing mechanism of human natural killer cells against hepatocellular carcinoma cell lines HepG2 and Hep3B

Purpose Unlike normal hepatocytes, most hepatocellular carcinomas (HCCs) are quite resistant to death receptor-mediated apoptosis when the cell surface death receptor is cross linked with either agonistic antibodies or soluble death ligand proteins in vitro. The resistance might play an essential role in the escape from the host immune surveillance; however, it has not been directly demonstrated that HCCs are actually resistant to natural killer (NK) cell-mediated death. Therefore, this study investigated the molecular mechanism of NK cell-mediated cytotoxicity against the HCCs, HepG2, and Hep3B, using two distinct cytotoxic assays: a 4-h ⁵¹Cr-release assay and a 2-h [³H] thymidine release assay which selectively measures the extent of necrotic and apoptotic target cell death, respectively.Methods Most of the target cells exhibited marked morphologic changes when they were co-incubated with the NK cells, and the NK cytotoxicity against these HCCs was comparable to that against K562, a NK-sensitive leukemia cell line, when the cytotoxicity was assessed by a 4-h ⁵¹Cr release assay.Results The NK cells also induced significant apoptotic cell death in the Hep3B targets, but not in the HepG2 targets, when the cytotoxicity was assessed by a 2-h [³H]-thymidine release assay. In agreement with these results, procaspase-3 was activated in the Hep3B targets, but not in the HepG2 targets. Interestingly, mildly fixed NK cells had no detectable activity in the 4-h ⁵¹Cr release assay against both HepG2 and Hep3B targets, while they were similarly effective as the untreated NK cells in the 2-h [³H]-thymidine release assay, suggesting that the level of apoptotic cell death of the Hep3B targets is granule independent and might be primarily mediated by the death ligands of the NK cells.Conclusion This study found that a tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/TRAIL receptor interaction is involved in the NK cell-mediated apoptotic death of the Hep3B targets, but a Fas/Fas ligand (FasL) interaction is not.     

N-acetylglycoside of oleanolic acid (aridanin) displays promising cytotoxicity towards human and animal cancer cells,inducing apoptotic,ferroptotic and necroptotic cell death

BackgroundThe discovery of novel phytochemicals represents a reasonable approach to fight malignancies, especially those which are resistant to standard chemotherapy.PurposeWe evaluated the cytotoxic potential of a naturally occurring N-acetylglycoside of oleanolic acid, aridanin, on 18 cancer cell lines, including sensitive and drug-resistant phenotypes mediated by P-glycoprotein, BCRP, p53 knockout, deletion-mutated EGFR, or BRAF mutations. Furthermore, metastasizing B16/F10 cells, HepG2 hepatocarcinoma and normal AML12 hepatocytes were investigated. The mechanisms of aridanin-induced cell death was further investigated.MethodsThe resazurin reduction assay (RRA) was applied to evaluate the cytotoxicity, autophagy, ferroptotic and necroptotic cell death. CCRF-CEM leukemia cells were used for all mechanistic studies. A caspase-Glo assay was applied to evaluate the caspase activities. Flow cytometry was applied for the analyses of cell cycle (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP; JC-1) and reactive oxygen species (ROS; H₂DCFH-DA).ResultsAridanin and doxorubicin (positive control) inhibited the proliferation of all cancer cell lines tested. The IC₅₀ values for aridanin varied from 3.18 µM (CCRF-CEM cells) to 9.56 µM (HepG2 cells). Aridanin had considerably lower IC₅₀ values than that of doxorubicin against multidrug-resistant CEM/ADR5000 cells and melanoma cell lines (MaMel-80a, Mel-2a, MV3, and SKMel-505). Aridanin induced apoptosis in CCRF-CEM cells through increase of ROS levels and MMP breakdown, and to a lesser extent via caspases activation. Aridanin also induced ferroptotic and necroptotic cell death.ConclusionThe present study opens good perpectives for the use of this phytochemical as an anticancer drug to combat multi-facorial resistance to established chemotherapeutics.     

PEGylated trimethylchitosan emulsomes conjugated to octreotide for targeted delivery of sorafenib to hepatocellular carcinoma cells of HepG2

Abstract

The current study aimed to develop PEGylated trimethyl chitosan (TMC) coated emulsomes (EMs) conjugated with octreotide for targeted delivery of sorafenib to hepatocellular carcinoma cells (HCC) of HepG2. Sorafenib loaded TMC coated EMs were prepared by the emulsion evaporation method and characterized concerning particle size, zeta potential, drug encapsulation efficiency, and in vitro drug release. Synthesized EMs were then conjugated to octreotide. The cytotoxicity of the targeted and non-targeted EMs was determined by cellular uptake and MTT assay on HepG2 cell. Cell cycle assay was also studied using flow cytometry. The results showed the optimized EMs had the particle size of 127?nm, zeta potential of ?5.41?mV, loading efficiency of 95%, and drug release efficiency of 62% within 52?h. Octreotide was attached efficiently to the surface of EMs as much as 71%. MTT assay and cellular uptake studies showed that targeted EMs had more cytotoxicity than free sorafenib and non-targeted EMs. Cell cycle analyses revealed that there was a significant more accumulation of targeted EMs treated HepG2 cells in the G1 phase than free sorafenib and non-targeted EMs. The results indicate that designed EMs may be promising for the treatment of hepatocellular carcinoma.     

In vitro impact of amino acid-derived bacterial metabolites on colonocyte mitochondrial activity,oxidative stress response and DNA integrity

Background4-hydroxyphenylacetic acid (HO-PAA) is produced by intestinal microbiota from L-tyrosine. High concentrations in human fecal water have been associated with cytotoxicity, urging us to test HO-PAA's effects on human colonocytes. We compared these effects with those of phenylacetic acid (PAA), phenol and acetaldehyde, also issued from amino acids fermentation.MethodsHT-29 Glc^−/+ human colonocytes were exposed for 24 h to metabolites at concentrations between 350 and 1000 μM for HO-PAA and PAA, 250-1500 μM for phenol and 25-500 μM for acetaldehyde. We evaluated metabolites'cytotoxicity with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and DNA quantification assays, reactive oxygen species (ROS) production with H2DCF-DA, and DNA damage with the comet assay. We measured cell oxygen consumption and mitochondrial complexes activity by polarography.ResultsAlthough HO-PAA displayed no cytotoxic effect on colonocytes, it decreased mitochondrial complex I activity and oxygen consumption. This was paralleled by an increase in ROS production and DNA alteration. Cells pretreatment with N-acetylcysteine, a ROS scavenger, decreased genotoxic effects of HO-PAA, indicating implication of oxidative stress in HO-PAA's genotoxicity. PAA and phenol did not reproduce these effects, but were cytotoxic towards colonocytes. Last, acetaldehyde displayed no effect in terms of cytotoxicity and mitochondrial metabolic activity, but increased DNA damage.ConclusionsSeveral bacterial metabolites produced from amino acids displayed deleterious effects on human colonocytes, in terms of genotoxicity (HO-PAA and acetaldehyde) or cytotoxicity (PAA and phenol).General significanceThis study helps understanding the consequences of intestinal microbiota's metabolic activity on the host since amino acids fermentation can lead to the formation of compounds toxic towards colonic epithelial cells.     

Synthesis,antitumor activity evaluation of some new N-aroyl-α,β-unsaturated piperidones with fluorescence

Novel N-aroyl-α,β-unsaturated piperidones, series 1, series 2 and series 3 (featuring 2-bromo-4,5-dimethoxybenzylidene, 4-dimethylaminobenzylidene and 4-trifluoromethylbenzylidene, respectively), were synthesized as candidate cytotoxins. Most of the compounds displayed potent cytotoxicity against the human neoplastic cell lines SK-BR-3, PG-BE1, NCI-H460, MIA PaCa-2 and SW1990 in vitro, and approximately 64% of the IC₅₀ values were lower than 5?μM. Among those tested, compound 1b of series 1, 3a, 3d and 3e of series 3 proved to be the most active. Importantly, 1b displayed marked inhibitory effects on tumor growth in vivo and had no apparent toxicity to mice; this was evaluated by a nude mouse PG-BE1 xenograft model. In addition, the fluorescent properties of compounds series 1–3 were investigated. The interesting fluorescence exhibited by these compounds could be useful for their visualization in tumor cells, permitting further studies on these α,β-unsaturated piperidones as candidates for novel fluorescent antitumor agents.