[Effects of Osthole on Apoptosis of HL-60 Cells Induced by TRAIL and Its Related Mechanism].

OBJECTIVE: To explore the effects of Osthole on apoptosis of HL-60 cells induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL) and its possible mechanism. METHODS: The proliferative inhibition of HL-60 cells treated with different concentrations of Osthole, TRAIL alone and Osthole combined with TRAIL was measured by MTT assay. The HL-60 cells were treated with Osthole, TRAIL alone and Osthole combined with TRAIL at the concentration

[Sensitization of human colon cancer HT-29 cells to TRAIL-induced apoptosis by gambognic acid].

To investigate the effects of gambognic acid (GA) on TRAIL-induced apoptosis of cancer cells, human colon HT-29 cancer cells were treated with GA to promote apoptosis. Inhibition of the cell proliferation was measured with MTT assay and cell apoptosis was detected with formation of DNA ladders in agarose gel electrophoresis, and activation of caspase activity. The content of cytosolic reactive oxygen species (ROS) was measured with flow cytometry. The activities of Caspase-3, -8, -9 were detected using spectrophotometric assay. The levels of c-FLIP, CHOP, DR4 and DR5 in cells were tested by Western blot. Combination of GA (1 µg · mL(-1)) and TRAIL (40 ng · mL(-1)) significantly reduced proliferation and increased apoptosis of HT-29 cells over those induced by each agent alone. Percentage of apoptotic cells was increased to 45.5%. GA markedly enhanced the intracellular ROS generation. Expression of CHOP, DR4 and DR5 was up-regulated to 7.38, 5.41, and 4.85 times of the control group, respectively. GA promoted activation of Caspase-3, -8, and -9 by TRAIL (P<0.05). Furthermore, the expression of anti-apoptotic protein c-FLIP was down-regulated to 0.22 ± 0.08 times of the control group. In conclusion, GA sensitizes HT-29 cells to TRAIL-induced apoptosis by promoting ROS-activated ERS pathways, up-regulating of DR4 and DR5, and inhibiting c-FLIP expression.

cDNA cloning, expression and bioactivity of porcine BAFF.

B cell activating factor belonging to the tumor necrosis factor (TNF) family (BAFF) is critical for B cell survival, maturation and T cell activation by acting through its three receptors, BAFF-R, BCMA and TACI. In the present study, a porcine BAFF cDNA, designated pBAFF, was cloned by RT-PCR and rapid amplification of cDNA ends (RACE) strategies. The full-length cDNA of pBAFF consists of 805bp with a 702bp open reading frame, encoding 233 amino acids. The deduced amino acid sequence contains a predicted transmembrane domain and a putative furin protease cleavage site corresponding to other identified BAFF homologues. The amino acid similarity between the functional soluble parts of pBAFF and human BAFF (hBAFF) or chicken BAFF (cBAFF) is 93% and 85%, respectively, with identity at the amino acid level was 88% and 76%, respectively. The characteristic of the three-cysteine residues of BAFF is conserved in pBAFF. RT-PCR showed that BAFF is expressed in many tissues in the pig, including spleen, liver, lung, heart, intestine, kidney, thymus and PBLs. Recombinant soluble pBAFF (psBAFF) fused with His(6) tag was efficiently expressed in Escherichia coli BL21 (DE3) and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blotting. In vitro, purified psBAFF co-stimulates the proliferation of not only porcine B cells but also human B cells. In addition, hsBAFF binds to porcine B cells and has a positive effect on their proliferation. These findings indicate pBAFF plays an important role in proliferation of porcine B cells and functional cross-reactivity occurs between porcine and human BAFF. In vitro expression of bioactive psBAFF provides the basis for further investigation of its potential to be used as an immunoadjuvant for enhancing vaccine efficacy and an immunotherapeutic in pig. It also provides the basis for investigations on the role of BAFF in this important domestic species and an animal model for human diseases.

[Cadmium induced apoptosis of HEK293 cells and its mitochondrial apoptosis pathway].

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.

Cloning, expression and bioactivity of duck BAFF.

B cell activating factor (BAFF) belonging to the TNF family is critical for B cell survival and maturation. In the present study, we identified a duck BAFF cDNA, named dBAFF, by RT-PCR and RACE strategies. The open reading frame (ORF) of this cDNA encodes a 288-amino acid protein containing a predicted transmembrane domain and a putative furin protease cleavage site like chicken BAFF (cBAFF), human BAFF (hBAFF) and mouse BAFF (mBAFF). The amino acid identity between biologically soluble dBAFF and cBAFF, hBAFF or mBAFF is 97, 78 and 71%, respectively. RT-PCR analysis showed the dBAFF gene is strongly expressed in the bursa of fabricius. Recombinant soluble dBAFF (dsBAFF) fused with NusA.tag was efficiently produced in Origami B (DE3) pLysS expression host strain. In vitro, purified dsBAFF was not only able to promote survival of bursa B cells, but also able to co-stimulate proliferation of mammalian B cells with anti-IgM. Furthermore, recombinant hsBAFF has a positive effect on duck bursa B cells survival. These findings indicate dBAFF plays an important role in survival and proliferation of duck B cells and because of its high conservation in the evolution, functional cross-reactivity exists between mammalian and duck BAFF.

Cadmium induces apoptosis in human embryonic kidney (HEK) 293 cells by caspase-dependent and -independent pathways acting on mitochondria.

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.

Cadmium-induced apoptosis in human normal liver L-02 cells by acting on mitochondria and regulating Ca(2+) signals.

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.

[Cloning, soluble expression and characterization of human sBCMA].

BCMA is one of the transmembrane receptors belonging to BAFF and APRIL. In order to identify the feasibility of sBCMA as decoy receptor and obtain active sBCMA for its structural and functional research, full length of hBCMA was amplified with total RNA from Raji cell line by RT-PCR, and the cDNA encoding the extracelluar soluble domain of hBCMA was inserted into pET43.1a(+) vector. The recombinant vector pET43.1a(+)-sBCMA was transformed into E. coli Origami B(DE3) pLyS which is helpful for disulfide bond construction of expression proteins. After IPTG induction, the recombinant protein was expressed as soluble fusion protein, sBCMA-NusA-His6, and identified by western blotting. Then the target protein was purified by Ni(+)-chelating Sepharose Fast Flow. The binding activity between recombinant sBCMA and BAFF was detected by ELISA. Also, Recombinant sBCMA inhibited proliferation of mouse B cell stimulating by rhsBAFF. It was proved that recombinant sBCMA has good bioactivity and the method to express those proteins rich in disulfide bond is feasible and effectual.