RESUMO
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.
Assuntos
Autofagia/efeitos dos fármacos , Cádmio/toxicidade , Hepatócitos/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteínas de Fusão de Membrana/genética , Proteínas de Fusão de Membrana/metabolismoRESUMO
The aim of this study was to construct a ribosome display library of single chain variable fragments (scFvs) associated with hepatocarcinoma and screen such a library for hepatocarcinoma-binding scFvs. mRNA was isolated from the spleens of mice immunized with hepatocellular carcinoma cell line HepG2. Heavy and k chain genes (VH and k) were amplified separately by RT-PCR, and an anti-HepG2 VH/k chain ribosome display library was constructed by assembling VH and k into the VH/k chain with a specially constructed linker by SOE-PCR. The VH/k chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. In order to isolate specific scFvs, recognizing HepG2 negative selection on a normal hepatocyte line WRL-68 was carried out before three rounds of positive selection on HepG2. After three rounds of panning, cell enzyme-linked immunosorbent assay (ELISA) showed that one of the scFvs had high affinity for the HepG2 cell and lower affinity for the WRL-68 cell. In this study, we successfully constructed a native ribosome display library. Such a library would prove useful for direct intact cell panning using ribosome display technology. The selected scFv had a potential value for hepatocarcinoma treatment.
Assuntos
Cadeias Pesadas de Imunoglobulinas/química , Neoplasias Hepáticas/metabolismo , Ribossomos/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Fragmentos de Imunoglobulinas , Neoplasias Hepáticas/química , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribossomos/químicaRESUMO
OBJECTIVE: To study the effects of cadmium on the structure and functions of mitochondria in hepatocytes. METHODS: Mitochondria were isolated from cultured human hepatocytes of the line WRL-68 and co-cultured with cadmium chloride (CdCl2) of the concentration of 1, 5, and 10 micromol/L, and WRL-68 cells not treated with CdCl2 (0 micromol/L) was used as control group. Cyclosporin A (CsA) was added into the culture medium. Mitochondrial permeability transition pore (MPTP) opening degree was tested by spectrophotometer. Morphologic changes of mitochondria were observed under transmission electron microscope. The activities of Na+ -K+ -ATPase, Ca2+ -Mg2+ -ATPase, LDH, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and the contents of malondialdehyde (MDA) were measured Mitochondria membrane potential (MMP) was monitored by spectrofluorimeter with fluorescence dye Rh-123. RESULTS: CdCl2 reduced the absorbance of mitochondria, signifying the opening of MPTP, concentration-dependently. The absorbance of mitochondria co-cultured with CsA and CdCl2 10 micromol/L was higher than that of the. CdCl2, 10 micromol/L group. Mild swelling was seen in the mitochondria treated with CdCl2. The MMP values of the CdCl2 5 and 10 micromol/L groups were significantly lower than that of the control group (P < 0.05, P < 0.01). The activity levels of ATPase, LDH, SOD, and GSH-Px in mitochondria decreased in the CdCl2 groups (all P < 0.05), and the contents of MDA increased in the CdCl2 groups compared with the control group. CONCLUSION: CdCl2 causes destruction of mitochondria structure, opening of MPTP, decrease of MMP, and changes of vitality of mitochondria enzymes that all play important roles in apoptosis of hepatocytes.
Assuntos
Cloreto de Cádmio/farmacologia , Hepatócitos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Linhagem Celular , Glutationa Peroxidase/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Malondialdeído/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Eletrônica , Mitocôndrias Hepáticas/fisiologia , Mitocôndrias Hepáticas/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Superóxido Dismutase/metabolismoRESUMO
HEK293 cell was chose to study the kidney damage of cadmium and to explore the significance of caspase 3,Bcl-2 and AIF (apoptosis inducing factor) in the apoptosis of cells induced by cadmium. Inhibition of the cell proliferation was measured by MTT assay. The structure of apoptotic cells was observed by light microscopy and electron microscopy; moreover, apoptotic cells were detected by DNA electrophoresis, flow cytometry and confocal laser microscopy. Furthermore,the expressions of Pro-caspase-3, Bcl-2 and the location of AIF in cells (mitochondria,cytoplasm or nuclei) were tested by western blot and immunofluorescence assay. CdCl2 exhibited anti-proliferative activity in dosage and time-dependent manner. DNA ladders of HEK293 cells were showed on agarose gel electrophoresis and the fragments of DNA were integral of 180-200 bp. 6-9 hours after 30 micromol/L CdCl2 treatment,DNA ladders were distinct. However, mistiness DNA ladder or smear was found when HEK293 cells were treated with CdCl2 on higher concentration or treated longer. It suggests that necrosis may happen, and flow cytometry results confirmed it. Morphological examination showed cell shrinkage, chromosomal condensation, karyotheca margination, nucleus cracking, vacuoles formed in cytoplasm and the presence of apoptotic bodies. At the same time,mitochondrial membrane potential (MMP) decreased, and the expression of Pro-caspase-3, Bcl-2 were decreased in time-dependent manner. Furthermore, AIF was released from mitochondria,and then traveled to nuclei. It suggests that CdCl2 may induce the apoptosis of HEK293 cells involving mitochondrial disruption including AIF migration and Cyt c release through both caspase-independent and -dependent pathways, and Bcl-2 and Caspase-3 are important factors which participate in the processes.
Assuntos
Apoptose/efeitos dos fármacos , Cádmio/farmacologia , Mitocôndrias/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular , Citometria de Fluxo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Confocal , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Cadmium is a well-known toxic compound for the liver. It has been demonstrated to induce hepatotoxicity partly via apoptosis, but no uniform mechanism of apoptosis has so far been proposed. This study was first to determine whether cadmium-induced apoptosis in L-02 cells, second to observe the mechanism of cadmium-induced apoptosis. Studies of morphology, DNA fragmentation and apoptotic rate demonstrated that 60µM cadmium induced apoptosis with strong effects on cell viability. A concomitant time-dependent decrease of Bcl-2 and mitochondrial transmembrane potential (ΔΨ(m)) was observed. Subsequently, increase of caspase-3 activity and release of mitochondrial AIF were detected. However, cell pretreatment with a broad-specificity caspase inhibitor (Z-Asp) did not abolish apoptosis. These data demonstrated that the apoptotic events involved a mitochondria-mediated apoptotic pathway but not necessarily caspase-dependent signaling. On the other hand, intracellular free Ca(2+) concentration ([Ca(2+)](i)) of cadmium-exposed cells had significant increases and the Bapta-AM, a well-known calcium chelator, pretreatment partially blocked cadmium-induced apoptosis, indicating that the elevation of [Ca(2+)](i) may play an important role in the apoptosis. Together, these results support the notion that cadmium-induced hepatotoxicity is comparable to effects in L-02 by inducing apoptotic pathways on the basis of acting on mitochondria and regulating Ca(2+) signals.
RESUMO
Cadmium (Cd) is a well-known toxic compound for the kidney in vivo and in vitro. It has been demonstrated to induce nephrotoxicity via in part by apoptotic cell death, but the precise mechanism is still unclear. Therefore, we have studied the effects of Cd on HEK 293 cells and investigated the mechanisms of Cd-induced apoptosis. Studies of morphology and oligonucleosomal DNA fragmentation demonstrated that 30-60 microM Cd induced apoptosis as early as 6-9h with strong effects on MTT activity, whereas 120 microM Cd revealed mainly necrosis, and the result of flow cytometry confirmed it. A concomitant time-dependent decrease of mitochondrial transmembrane potential (DeltaPsi(m)) and Bcl-2 expression was observed, subsequently, release of cytochrome c (Cyt c) and activation of caspase-3 were detected, suggesting a caspase-dependent pathway. Meanwhile, mitochondrial AIF was released to cytoplasm and nucleus, suggesting a caspase-independent pathway. Furthermore, when cells were transfected with pcDNA3/Bcl-2 before exposed to CdCl(2), alleviated apoptosis was assessed by part of the apoptotic features in this study. Taken together, our results showed that CdCl(2) caused time- and dose-dependent apoptosis or even necrosis in HEK 293 cells depending on the exposure conditions. The apoptotic events may involve mitochondrial disruption including both caspase-dependent and -independent pathways.