Normal Human Liver Cell Line Research Articles

Death receptor and mitochondria-mediated hepatocyte apoptosis underlies liver dysfunction in rats exposed to organic pollutants from drinking water.

Persistent organic pollutants in drinking water impose a substantial risk to the health of human beings, but the evidence for liver toxic effect and the underlying mechanism is scarce. This study aimed to examine the liver toxicity and elucidate the molecular mechanism of organic pollutants in drinking water in normal human liver cell line L02 cells and rats. The data showed that organic extraction from drinking water remarkably impaired rat liver function, evident from the increase in the serum level of alanine aminotransferase, aspartate aminotransferase, and cholinesterase, and decrease in the serum level of total protein and albumin. Organic extraction dose-dependently induced apoptotic cell death in rat liver and L02 cells. Administration of rats with organic extraction promoted death receptor signaling pathway through the increase in gene and protein expression level of Fas and FasL. Treatment of rats with organic extraction also induced mitochondria-mediated apoptosis via increasing the expression level of proapoptotic protein, Bax, but decreasing the expression level of antiapoptotic protein, Bcl-2, resulting in an upregulation of cytochrome c and activation of caspase cascade at both transcriptional and post-transcriptional levels. Moreover, organic extraction enhanced rat liver glutathione S-transferases activity and reactive oxygen species generation, and upregulated aryl hydrocarbon receptor and glutathione S-transferase A1 at both transcriptional and translational levels. Collectively, the results indicate that organic extraction from drinking water impairs liver function, with the involvement of death receptor and mitochondria-mediated apoptosis in rats. The results provide evidence and molecular mechanisms for organic pollutants in drinking water-induced liver dysfunction, which may help prevent and treat organic extraction-induced liver injury.

Effects of Ras homolog gene family, member C gene silencing combined with rapamycin on hepatocellular carcinoma cell growth.

The aim of the present study was to investigate the combined effects of inhibiting the Ras homolog gene family, member C (RhoC)/Rho kinase and phosphoinositide 3 kinase/Akt/mammalian target of rapamycin (mTOR) pathways on hepatocellular carcinoma cell growth. The RhoC gene was silenced by RNA interference (RNAi) and mTOR was inhibited by rapamycin (RAPA). Subsequently, an MTT assay for cell growth detection, western blot analysis for gene expression analysis, silver nitrate staining for cell proliferation, Wright's staining for analysis of the apoptotic rate analysis, soft agar clonogenic assay for the determination of cell growth characteristics and a Transwell assay for cell migration were performed. RhoC expression in hepatoma cell lines was lower than that in the HL7702 normal human liver cell line. The level of cell proliferation in the RNAi + RAPA group was lower than that in the RNAi, RAPA and Scramble groups. The levels of cyclin-dependent kinase 2 in the RNAi + RAPA group were lower than those in the other groups, while the levels of P16 in the RNAi + RAPA group were higher than those in the other experimental groups. No significant difference was found between the RNAi + RAPA and the normal HL7702 group. The number of silver nitrate-stained particles was reduced in the RNAi + RAPA group compared with that in the other groups. No significant difference was found between the RNAi + RAPA and HL7702 groups. Wright's staining for apoptosis demonstrated that apoptosis in the Scramble group was rare, while the RAPA and RNAi groups contained a large number of apoptotic cells, which displayed nuclear condensation, fragmentation, deepened staining, as well as a wrinkled membrane. B-cell lymphoma-2 (Bcl-2) expression in the RNAi + RAPA group was lower than that in the other groups, while the gene expression of Bcl-2-associated X protein in the RNAi + RAPA group was increased compared with that in the other groups. No cell colony formation was observed in the soft agar cloning experiment in the RNAi + RAPA and HL7702 group, while in the other groups, visible cell clones appeared. In the Transwell assay the number of migrated cells in the RNAi + RAPA group was lower than that in the other groups. The gene expression of matrix metalloproteinase (MMP)2, MMP-9 and vascular endothelial growth factor in the RNAi + RAPA group was lower than that in the other experimental groups. In conclusion, RhoC gene silencing combined with RAPA was able to significantly inhibit the growth of hepatocellular carcinoma cells.

Reduced ubiquitin-specific protease 9X expression induced by RNA interference inhibits the bioactivity of hepatocellular carcinoma cells.

Ubiquitin-specific protease 9X (USP9X) is crucial in many tumor types, but not in hepatocellular carcinoma (HCC). The current study aimed to examine the effects of RNA interference on USP9X expression, and subsequently on the bioactivity of HCC SMMC7721 and HepG2 cells. The protein expression of USP9X in SMMC7721, HepG2 and normal human liver cell line L02 at the cellular level was determined by western blot analysis; USP9X was knocked down by small interfering RNA (siRNA) in HCC SMMC7721 and HepG2 cells. In vitro cell viability was assessed by MTT assay, apoptosis was determined by flow cytometry (FCM) and cell migration was evaluated by Transwell assays. The protein expression of USP9X in SMMC7721 and HepG2 were both significantly higher than that in L02 (P<0.01). The results of western blot demonstrated that the USP9X-siRNA could efficiently inhibit USP9X expression when compared with that of the negative control (NC) group (P<0.01) and MTT assay demonstrated that cell proliferation in USP9X-blocked cells was significantly reduced when compared with that of the NC group (P<0.01). The results of FCM revealed that apoptosis was significantly increased in USP9X-blocked cells when compared with that of the NC group (P<0.01). The results of transwell assay showed that cell migration was significantly inhibited in USP9X-blocked cells when compared with that of the NC group (P<0.01). These results show that expression of USP9X is upregulated in hepatoma cells SMMC7721 and HepG2, and that downregulating USP9X by siRNA may induce cell apoptosis, inhibit cell growth and cell migration in the HCC SMMC7721 and HepG2 cell lines. USP9X may therefore be a potential target for HCC treatment and early detection.

Retraction note: Ferroportin in the progression and prognosis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor in men and the seventh in women and understanding the molecular mechanisms of HCC and establishing more effective therapies are critical and urgent issues. Our objective was to study the expression of ferroportin in hepatocellular carcinoma (HCC) tissue samples and the relationship between ferroportin expression and HCC characteristics. Sixty HCC tissues and their corresponding para-cancer liver tissues (PCLT) were obtained from sixty HCC patients who had undergone hepatectomy in the Second Xiangya Hospital of Central South University. Ten normal liver tissue samples were also obtained as a control. Immunohistochemistry (IHC) was performed to analyze the ferroportin expression in HCC, and the relationship between ferroportin expression and HCC clinical pathological characteristics also was analyzed. For the evaluation of IHC results, the comprehensive scoring criteria were met according to the staining intensity and the number of positive staining cells. Western blotting was performed to detect the expression level of ferroportin in HCC cell lines. Ferroportin expression in HCC tissue was significantly lower compared to PCLT and normal liver tissue (P <0.05). Moreover, ferroportin expression was related to liver cancer cell de-differentiation, the severity degree in TNM staging, Edmondson-Steiner grading, intrahepatic metastasis and portal vein invasion. In addition, high expression of ferroportin was observed in normal human liver cell lines L02 and HL7702, whereas weak positive expression and even negative expression of ferroportin were observed in HCC cell lines FOCUS, MHCC-97H, HepG2 and SMMC-7721. Furthermore, among the four kinds of HCC cell lines, the expression level of ferroportin was the lowest in MHCC-97H cells. Ferroportin expression level declines along with the progression of liver cancer, suggesting that the reduction of ferroportin may serve as an important marker for poor HCC prognosis and as a new therapeutic target.

Investigation of trichloroethylene-induced effects on subcellular proteomes in L-02 hepatic cells

To put the insight into the trichloroethylene (TCE)-induced effect on the differential expression of subcellular proteins in human normal liver cell line (L-02). The membrane proteins and nuclear proteins of TCE-treated (8.0 mmol/L) group and controls were extracted by subcellular proteome extraction kit, respectively. The TCE-induced differentially expressions were analyzed by a two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization tandem time-of-flight spectrometry (MALDI-TOF-MS). Bioinformatics analysis was used to reveal the biological processes and predict transmembrane domains of differential expressed proteins. The expression of ATP synthase subunit beta (ATP5B), heterogeneous nuclear ribonucleoprotein H2 (hnRNP H2) and far up steam element-binding protein 1 (FUBP1) were measured under TCE treatment by Western blot. After TCE treatment for 24 h in L-02 cells, 14 membrane proteins and 18 nuclear proteins were identified as differential expression. After treated with TCE in concentrations of 0, 2.0, 4.0 and 8.0 mmol/L for 24 h, the relative levels of ATP5B expression were 1.00±0.03, 1.21±0.14, 1.25±0.12 and 1.48±0.17 (F = 8.51, P = 0.007), the relative levels of hnRNP H2 expression were 1.00±0.09, 1.22±0.15, 1.43±0.21, 1.53±0.17 (F = 6.57, P = 0.015), respectively; the relative levels of FUBP1 expression were 1.00±0.11, 0.91±0.07, 0.73±0.04 and 0.67±0.03 (F = 15.81, P = 0.001), respectively, which were consistent with the results in proteomics. The bioinformatics analysis showed that the most dominant biological process were involved in RNA processing (10 proteins, P = 2.46×10(-6)), especially in RNA splicing (9 proteins, P = 1.77×10(-7)). The exposure of TCE could alter the expression of membrane proteins and nuclear proteins in L-02 cells. These abnormal expressed proteins involved in RNA splicing would provide novel clues for further understanding of TCE-induced hepatotoxicity.

Carboxylation of multiwalled carbon nanotube attenuated the cytotoxicity by limiting the oxidative stress initiated cell membrane integrity damage, cell cycle arrestment, and death receptor mediated apoptotic pathway.

In this study, the effects of carboxylated multiwalled carbon nanotubes (MWCNTs-COOH) on human normal liver cell line L02 was compared with that of pristine multiwalled carbon nanotubes (p-MWCNTs). It was shown that compared with MWCNTs-COOH, p-MWCNTs induced apoptosis, reduced the level of intracellular antioxidant glutathione more significantly, and caused severer cell membrane damage as demonstrated by lactate dehydrogenase leakage. Cell cycles were arrested by both MWCNTs, while p-MWCNTs induced higher ratio of G0/G1 phase arrestment as compared with MWCNTs-COOH. Caspase-8 was also activated after both MWCNTs exposure, indicating extrinsic apoptotic pathway was involved in the apoptosis induced by MWCNTs exposure, more importantly, MWCNTs-COOH significantly reduced the activation of caspase-8 as compared with p-MWCNTs. All these results suggested that MWCNTs-COOH might be safer for in vivo application as compared with p-MWCNTs.

The relationship between Cd-induced autophagy and lysosomal activation in WRL-68 cells.

This study shows that Cd induces autophagy in the human's embryonic normal liver cell line (WRL-68). The expression of LC3B-II and the mature cathepsin L were analyzed by Western blotting. The autophagosomes and lysosomes were directly visualized by electron microscopy and confocal microscopy analysis in Cd-exposed WRL-68 cells. In this study, we first found that autophagy induced the activation of lysosomal function in WRL-68 cells. The lysosomal activation was markedly decreased when the cells were co-treated with 3-MA (an inhibitor of autophagy). Secondly, we provided the evidence that the activation of lysosomal function depended on autophagosome-lysosome fusion. The colocalization of lysosome-associated membrane protein-2 (LAMP2) and GFP-LC3 was significantly reduced, when they were treated with thapsigargin (an inhibitor of autophagosome-lysosome fusion). We demonstrated that deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, which suggests that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Thirdly, we provided evidence that the activation of lysosomal function was associated with lysosomal acid. We investigated the relationship between autophagosome-lysosome fusion and pH in acidic compartments by visualizing fusion process in WRL-68 cells. This suggests that increasing pH in acidic compartments in WRL-68 cells inhibits the autophagosome-lysosome fusion. Finally, we found that the activation of lysosomal function was associated with Ca(2+) stores and the intracellular Ca(2+) channels or pumps were possibly pH-dependent.

Relationship between CCL20/CCR6/Th17 axis and vascular invasion and metastasis in patients with primary hepatocellular carcinoma

To investigate the role of CCL20/CCR6/Th17 axis in vascular invasion and metastasis of primary hepatocellular carcinoma (HCC). Expression levels of CCL20 mRNA in the normal human liver cell line L-02, and human hepatocellular carcinoma cell lines Hep3B, Huh7 and HepG2 were quantified by using SYBR green real time PCR. CCL20 secretions from these cell lines were quantified by using ELISA. The chemotactic effect of HCC cell line Hep3B on human peripheral blood mononuclear cells was determined by using transwell chemotaxis assay. Pre-therapy serum levels of IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-17, IL-23, IFN-γ, TNF-α and CCL20 in 93 patients with HCC were measured by using 9-plex array and ELISA. All the patients were chronic hepatitis B virus associated HCC, and 51 cases were those with vascular invasion and metastasis (metastasis group) and 42 cases were not (non-metastasis group). CCL20 and CCR6 mRNA expressions in the HCC and tumor-adjacent tissues were determined by using SYBR Green real time PCR in 41 patients, among them, 20 cases were from the group of patients with metastasis and 21 cases were from the group of patients without metastasis. The CCL20 expression was further determined by immunohistochemistry. The HCC cell lines expressed and secreted higher amount of CCL20, which effectively recruited CCR6(+) T cells. Pre-therapy serum levels of CCL20 in 93 HCC patients were (38.2 ± 28.4)pg/ml, significantly increased than those with benign hepatic hemangiomas [(7.8 ± 17.8)pg/ml, P < 0.01]. In addition, the serum levels of CCL20 were positively correlated with the tumor diameters in HCC patients (r = 0.32, P = 0.0018). CCL20 was dominantly expressed in the cytoplasm in HCC cells, and it was also expressed by some infiltrating immune cells. The mRNA expression levels of CCL20 of the tumor tissues were significantly higher than that in the tumor-adjacent tissues (P < 0.05). Multivariate logistic regression analysis showed that serum levels of IL-17 and CCL20 were independent risk factors of metastasis in HCC patients (P < 0.05 for both). CCL20 mRNA showed no statistically significant differences between patients with metastasis and without metastasis in both tumor tissues and tumor-adjacent tissues (P > 0.05 for both). But the patients with metastasis showed significantly higher expressions of CCR6 both in their tumor [5.75 (1.79, 19.13)]and tumor-adjacent tissues [7.99 (4.49, 19.54)] than those with non-metastasis [1.69 (0.76, 2.87) and 3.58 (1.84, 4.32), P < 0.05 for both]. CCL20/CCR6/Th17 axis may promote vascular invasion and metastasis hepatocellular carcinoma.

Cytotoxicity and anticancer studies of Bacillus cereus and Bacillus pumilus metabolites targeting human cancer cells

The metabolites of bacteria Bacillus cereus and Bacillus pumilus isolated from soil samples in Shimoga region, Karnataka (India) were tested for cytotoxicity and anticancer properties. The various solvent extract fractions obtained from the metabolites of the two bacteria were tested for their cytotoxicity against normal human liver cell lines and 2 cancer cell lines by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay. The two fractions obtained from B. cereus showed high cytotoxicity. These two fractions were further screened for anticancer activity by nuclear staining studies and DNA fragmentation analysis. Both the fractions demonstrated significant activity by membrane blebbing during nuclear staining and caused the damage the DNA patterns during DNA fragmentation analysis. On the other hand, the metabolites of B. pumilus revealed toxic effect against cancer cells as well as normal ones.

Role of the exogenous HCV core protein in the interaction of human hepatocyte proliferation and macrophage sub-populations.

BackgroundThe core protein of hepatitis C virus (HCV) is found in the cytoplasm and nuclei of infected cells, including hepatocytes and other cells in the liver. The core protein could be secreted as well. Resident liver macrophages are dependent on the tissue micro-environment and external stimuli to differentiate M1 and M2 hypotypes with distinct functions, and increased expression of the nuclear transcription factor STAT3 was seen in M2-polarized macrophages. In contrast to proinflammatory M1 macrophages, M2 macrophages serve beneficial roles in chronic inflammation, immunosuppression, and tumorigenesis.MethodsMonocyte-derived human macrophage line (mTHP-1) was treated with the exogenous HCV core protein. Next, the mTHP-1 culture supernatant or cell pellets were added to culture media of normal human liver cell line (L02).ResultsOnly the culture supernatant stimulated L02 cells proliferation, which was associated with phosphorylated ERK expression. Core protein activated mTHP-1 cells showed enhanced pro- and anti-inflammatory cytokines secretion, which was accompanied by high expression of phosphorylated NF-κB105 and NF-κB65. However, phosphorylated STAT1, and STAT3, which are normally associated with M1 and M2 macrophage polarization, and cell surface expression of CD206, CD14, CD16, and CD86, were unaltered. A transwell co-culture system showed that only in mTHP-1 co-cultured with L02 in the presence of exogenous core protein, were higher levels of phosphorylated STAT3 and CD206 seen.ConclusionsWe showed L02 cells proliferation was accelerated by the culture supernatant of mTHP-1 cells treated with the exogenous HCV core protein. The exogenous core protein mediated the interaction between macrophages and hepatocytes in co-culture, which enhanced the expression of phosphorylated STAT3 and CD206 in macrophages.

TPX2 knockdown suppressed hepatocellular carcinoma cell invasion via inactivating AKT signaling and inhibiting MMP2 and MMP9 expression.

Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a nuclear proliferation-related protein that plays a critical role in the formation of mitotic spindle. High expression of TPX2 has been observed in several types of tumors. However, the role of TPX2 in hepatocellular carcinoma (HCC) remains unclear. Our study aimed to investigate the effect of TPX2 on HCC cell invasion. The immortalized normal human liver cell line L02 and six HCC cell lines including SMMC-7721, BEL-7402, Huh-7, HepG2, Hep3B and SKHep1 were subjected to qRT-PCR and western blot for TPX2 mRNA and protein, respectively. Furthermore, TPX2 small interfering RNA (siRNA) was used to knock down TPX2 expression in SMMC-7721 and HepG2 cells. Cell proliferation and invasion were determined by MTT and transwell assays. Otherwise, expression of p-AKT, MMP2 and MMP9 were evaluated by western blot in SMMC-7721 cells. The expression of TPX2 in HCC cell lines was markedly higher than that in normal human liver cell line. TPX2 knockdown using a specific TPX2-siRNA reduced the number of invaded cells and inhibited cell proliferation in SMMC-7721 and HepG2 cells. Furthermore, TPX2 knockdown resulted in inactivation of AKT signaling and down-regulation of MMP2 and MMP9 expression in SMMC-7721 cells. Our study identified that TPX2 might contribute to tumor cell invasion through activating AKT signaling and subsequently increasing MMP2 and MMP9 in HCC.

The role of N-acetyltransferase 8 in mesenchymal stem cell-based therapy for liver ischemia/reperfusion injury in rats.

ObjectiveTo evaluate the impact of mesenchymal stem cells (MSCs) against hepatic I/R injury and explore the role of N-acetyltransferase 8 (NAT8) in the process.MethodsWe investigated the potential of injected MSCs systemically via the tail vein in healing injuried liver of the SD rat model of 70% hepatic I/R injury by measuring the biochemical and pathologic alterations. Subsequently, we evaluated the expression levels of NAT8 by western blotting in vivo. Concurrently, hydrogen peroxide (H2O2)-induced apoptosis in the human normal liver cell line L02 was performed in vitro to evaluate the protective effects of MSC conditioned medium (MSC-CM) on L02 cells. In addition, we downregulated and upregulated NAT8 expression in L02 cells and induced apoptosis by using H2O2 to study the protective role of NAT8.ResultsMSCs implantation led to a significant reduced liver enzyme levels, an advanced protection in the histopathological findings of the acutely injured liver and a significantly lower percentage of TUNEL-positive cells, which were increased after I/R injury. In vitro assays, MSC-CM inhibited hepatocyte apoptosis induced by H2O2. Moreover, overexpression or downregulation of NAT8 prevented or aggravated hepatocyte apoptosis induced by H2O2, respectively.ConclusionsMSC transplantation provides support to the I/R-injured liver by inhibiting hepatocellular apoptosis and stimulating NAT8 regeneration.

Dihydromyricetin induces apoptosis and inhibits proliferation in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a life-threatening disease that is known to exhibit a poor prognosis. Therefore, it is important to identify an effective drug therapy for the treatment of HCC. Dihydromyricetin (DHM) is a flavonoid compound, isolated from the classical Chinese herb Ampelopsis grossedentata, which exhibits multiple pharmacological activities, including anticancer effects. In this study, the anticancer effect of DHM was investigated in nine different types of HCC cell lines via cell proliferation and immunoassays, as well as apoptosis detection. Two immortalized normal human liver cell lines were utilized to determine hepatotoxicity. The results revealed that DHM significantly inhibited cell proliferation and induced cell apoptosis in the HCC cell lines. However, DHM exhibited no cytotoxicity to normal human hepatic cell lines. Furthermore, it was found that DHM induced cell apoptosis in a p53-dependent manner. DHM upregulated p53 expression, and the upregulation of p53 increased the levels of the cleaved caspase-3 protein, directly inducing cell apoptosis. These results indicate that DHM is a promising candidate for the treatment of HCC.

A novel fluorescent “turn-on” chemosensor for nanomolar detection of Fe(III) from aqueous solution and its application in living cells imaging

An electronically active and spectral sensitive fluorescent “turn-on” chemosensor (BTP-1) based on the benzo-thiazolo-pyrimidine unit was designed and synthesized for the highly selective and sensitive detection of Fe3+ from aqueous medium. With Fe3+, the sensor BTP-1 showed a remarkable fluorescence enhancement at 554nm (λex=314nm) due to the inhibition of photo-induced electron transfer. The sensor formed a host-guest complex in 1:1 stoichiometry with the detection limit down to 0.74nM. Further, the sensor was successfully utilized for the qualitative and quantitative intracellular detection of Fe3+ in two liver cell lines i.e., HepG2 cells (human hepatocellular liver carcinoma cell line) and HL-7701 cells (human normal liver cell line) by a confocal imaging technique.

Synthesis, crystal structure, cytotoxicity and cell apoptosis induction of a copper(II)-based Schiff base complex

A new copper(II) complex, [CuII(ClQP)(NO3)(H2O)] (1), bearing with 4-Chloro-2-(quinolin-8-yliminomethyl)-phenol (HClQP) as Schiff base ligand, was synthesized and structurally characterized by IR, elemental analysis, ESI-MS and single crystal X-ray diffraction analysis. The crystal structure revealed that complex 1 was five coordinated by one N,N,O-tridentated ClQP, one H2O and one nitrate anion, respectively, to form a pyramidal coordinating geometry. The antitumor activity of complex 1 against HepG2, BEL-7404, MGC80-3 and NCI-H460 tumor cell lines were screened by MTT assay. The IC50 values were in the range of 6.53–23.62μM, in which HepG2 cell line showed the highest sensitivity to complex 1. It is worth noting that complex 1 did not show cytotoxicity to human normal liver cell line HL-7702, suggesting its cytotoxic selectivity on these tumor cell lines. Towards the most sensitive HepG2 cell line, complex 1 significantly induced S phase cell cycle arrest, probably due to its intercalative binding with DNA based on its rigid planar structure as well as the planar aromatic groups of HClQP ligand. Furthermore, the HepG2 cells treated with 1 showed typical cell apoptosis in dose-dependent manner visualized by Hoechst 33258 staining. And the cell apoptosis in the HepG2 cells induced by 1 was further confirmed by the loss of mitochondrial membrane potential visualized by JC-1 staining in cells. The detailed flow cytometric assay revealed that complex 1 could up-regulate the expression of the caspase-3. From these results, it can be concluded that complex 1 induced the mitochondrion-mediated cell apoptosis by caspase-3 activation, most probably due to the S phase cell cycle arrest in HepG2 cells owing to the DNA binding affinity of 1via intercalative binding mode.

Antitumor properties and mechanisms of mitochondria-targeted Ag(I) and Au(I) complexes containing N-heterocyclic carbenes derived from cyclophanes.

Metal/N-heterocyclic carbene (NHC) complexes hold great opportunities for the development of novel antitumor metallodrugs. Herein, four Ag(I) and Au(I) complexes containing NHCs derived from cyclophanes have been prepared and characterized as potential anticancer agents. These complexes show higher cytotoxicity than cisplatin against several cancer cell lines including a cisplatin-resistant cell line. The Au(I) complexes display higher anticancer activity than the corresponding Ag(I) complexes. Moreover, all the complexes are less cytotoxic than cisplatin on the normal human liver cell line LO2. Intracellular distribution studies show that these complexes are selectively localized in the mitochondria, especially for the Au(I) complexes. Interestingly, although both Ag(I) and Au(I) complexes can influence mitochondrial integrity, they induce cancer cell death through different mechanisms. The Ag(I) complexes mainly induce caspase- and reactive oxygen species (ROS)-independent early apoptosis. However, Au(I) complexes induce both early apoptosis and late apoptosis through caspase- and ROS-dependent pathways. These studies may open up an avenue for a better understanding of the impact of metal centers on antitumor potentialities and mechanisms.

Mono(2-ethylhexyl) phthalate induces apoptosis in p53-silenced L02 cells via activation of both mitochondrial and death receptor pathways

Mono(2-ethylhexyl) phthalate (MEHP) is one of the main metabolites of di(2-ethylhexyl) phthalate. The evidence shows that DEHP may exert its toxic effects primarily via MEHP, which is 10-fold more potent than its parent compound in toxicity in vitro. MEHP-induced apoptosis is mediated by either p53-dependent or -independent pathway. However, the detailed mechanism of its toxicity remains unclear. In this study, immortalized normal human liver cell line L02 was chosen, as an in vitro model of nonmalignant liver, to elucidate the role of p53 in MEHP-induced apoptosis. The cells were treated with MEHP (6.25, 12.50, 25.00, 50.00, and 100.00 μM) for 24 and 36 h, then small interfering RNA (siRNA) was used to specifically silence p53 gene of L02 cells. The results indicated that MEHP caused oxidative DNA damage and apoptosis in L02 cells were associated with the p53 signaling pathway. Further study found that MEHP (50.00 and 100.00 μM) induced apoptosis in p53-silenced L02 cells, along with the up-regulations of Fas and FasL proteins as well as increased the Bax/Bcl-2 ratio and Caspase 3, 8, and 9 activities. Additionally, both FasL inhibitor (AF-016) and Caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp- fluoromethylketone (Z-VAD-FMK) could prevent the cell apoptosis induced by MEHP. The findings suggested that MEHP-induced apoptosis in L02 cells involving a Caspases-mediated mitochondrial signaling pathway and/or death receptor pathway. p53 was not absolutely necessary for MEHP-induced L02 cell apoptosis.

Hyperphosphorylation of microfilament‐associated proteins is involved in microcystin‐LR‐induced toxicity in HL7702 cells

Microcystin-LR (MC-LR) has been regarded as a hepatotoxin, which can cause cytoskeletal reorganization, especially of the actin filaments. However, the underlying mechanisms remain unclear. In this study, whether MC-LR could induce microfilaments disruption was verified in the normal human liver cell line HL7702; and then the transcription, translation, and phosphorylation levels of major microfilament-associated proteins were measured; finally, the underlying mechanisms was investigated. After treatment with MC-LR, the actin filaments lost their characteristic filamentous organization in the cells, demonstrating increased actin depolymerization. The mRNA and protein levels of ezrin, vasodilator-stimulated phosphoprotein (VASP), actin-related protein2/3, and cofilin remained unchanged. However, the phosphorylation levels of ezrin and VASP were increased, when treated with 10 μM MC-LR. Moreover, P38 and ERK1/2 were involved in MC-LR-induced hyperphosphorylation of microfilament-associated proteins. In summary, this study demonstrates that MC-LR can cause disruption of actin filaments in HL7702 cells due to MC-LR-induced mitogen-activated protein kinase pathway activation and hyperphosphorylation of different types of microfilament-associated proteins.

Anti-Tumor Effects of an Oncolytic Adenovirus Expressing Hemagglutinin-Neuraminidase of Newcastle Disease Virus in Vitro and in Vivo

Oncolytic virotherapy has been an attractive drug platform for targeted therapy of cancer over the past few years. Viral vectors can be used to target and lyse cancer cells, but achieving good efficacy and specificity with this treatment approach is a major challenge. Here, we assessed the ability of a novel dual-specific anti-tumor oncolytic adenovirus, expressing the hemagglutinin-neuraminidase (HN) gene from the Newcastle disease virus under the human telomerase reverse transcriptase (hTERT) promoter (Ad-hTERTp-E1a-HN), to inhibit esophageal cancer EC-109 cells in culture and to reduce tumor burden in xenografted BALB/c nude mice. In vitro, infection with Ad-hTERT-E1a-HN could inhibit the growth of EC-109 cells significantly and also protect normal human liver cell line L02 from growth suppression in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Ad-hTERT-E1a-HN also effectively and selectively decreased the sialic acid level on EC-109 cells, but not on L02 cells. Furthermore, Ad-hTERT-E1a-HN was shown to induce the apoptosis pathway via acridine orange and ethidium bromide staining (AO/EB staining), increase reactive oxygen species (ROS), reduce mitochondrial membrane potential and release cytochrome c. In vivo, xenografted BALB/c nude mice were treated via intratumoral or intravenous injections of Ad-hTERT-E1a-HN. Although both treatments showed an obvious suppression in tumor volume, only Ad-hTERT-E1a-HN delivered via intratumoral injection elicited a complete response to treatment. These results reinforced previous findings and highlighted the potential therapeutic application of Ad-hTERT-E1a-HN for treatment of esophageal cancer in clinical trials.

Impact Assessment of Cadmium Toxicity and Its Bioavailability in Human Cell Lines (Caco-2 and HL-7702)

Cadmium (Cd) is a widespread environmental toxic contaminant, which causes serious health-related problems. In this study, human intestinal cell line (Caco-2 cells) and normal human liver cell line (HL-7702 cells) were used to investigate the toxicity and bioavailability of Cd to both cell lines and to validate these cell lines as in vitro models for studying Cd accumulation and toxicity in human intestine and liver. Results showed that Cd uptake by both cell lines increased in a dose-dependent manner and its uptake by Caco-2 cells (720.15 µg mg−1 cell protein) was significantly higher than HL-7702 cells (229.01 µg mg−1 cell protein) at 10 mg L−1. A time- and dose-dependent effect of Cd on cytotoxicity assays (LDH release, MTT assay) was observed in both Cd-treated cell lines. The activities of antioxidant enzymes and differentiation markers (SOD, GPX, and AKP) of the HL-7702 cells were higher than those of Caco-2 cells, although both of them decreased significantly with raising Cd levels. The results from the present study indicate that Cd above a certain level inhibits cellular antioxidant activities and HL-7702 cells are more sensitive to Cd exposure than Caco-2 cells. However, Cd concentrations <0.5 mg L−1 pose no toxic effects on both cell lines.