Effects of oxidized low density lipoprotein and vitamin E on the levels of IL-6,IL-8 and TNF-α in cultured human umbilical vein endothelial cells

AIM:To investigate effects of OX-LDL and VitE on the levels of IL-6,IL-8 and TNF-α in human umbilical vein endothelial cells(HUVEC).METHODS: Human umbilical vein endothelial cells were obtained by in vitro culture. HUVEC treated with or without Vit E was incubated with OX-LDL, and the levels of IL-6, IL-8 and TNF-α were determined by enzyme-linked immunosorbent assy technique. RESULTS:50 μg/L,100 μg/L, 200 μg/L OX-LDL induced the release of IL-6,IL-8 and TNF-α by HUVEC in a dose-dependent manner. Compared with the control group , the levels of IL-6 and IL-8 were significantly increased at 6-12 h of stimulation with OX-LDL . Maximal levels of IL-6 and IL-8 occurred after 24-36 h, reaching a plateau maintained for at least 48 h. TNF-α rose after 2-6 h in HUVEC, and reached a maximum after 12 h. In contrast to IL-6 and IL-8, TNF-α declined after 48 h. However, when VitE (50 mg/L,100 mg/L,200 mg/L)was added, it can significant inhibited the release of IL-6, IL-8 and TNF-α in a dose-dependent manner, and after 48 h these cytokines have no diference between OX-LDL+VitE groups and OX-LDL groups. CONCLUSION: OX-LDL can obviously stimulate the production of IL-6,IL-8 and TNF-α in vascular endothelial cells, which can significantly be inhibited by VitE in a short time.     

Modulation of bacterial redox protein azurin induces human osteosarcoma cell apoptosis by Fas antigen and caspase-8

AIM: To determine the role of Fas antigen and caspase-8 in modulating apoptosis of osteosarcoma cells induced by bacterial redox protein azurin. METHODS: The human osteosarcoma cell line U2OS was treated with bacterial redox protein azurin (150 mg/L) for 6, 12, 24 and 48 h, respectively. Cell immunohistochemistry and quantitative image pattern analysis were applied for detecting the expression of Fas antigen. Caspase-8 activity was detected using caspase-8 fluorescent assay kit. The apoptotic rate was measured by FCM. RESULTS: Compared with the control group, the expression of Fas antigen and activity of caspase-8 significantly increased in U2OS cells treated with 150 mg/L azurin (P<0.01). There was positive correlation between the expression of Fas antigen and activity of caspase-8 (r=0.873, P<0.01). The increased Fas antigen expression had the function to transfer apoptotic signals and the anti-Fas antibody promoted azurin induced apoptosis through increasing Fas antigen expression. IETD-FMK blocked the activation of procaspase-8 and reduced apoptosis of U2OS cells in the presence of azurin or anti-Fas antibody. The apoptotic rates in azurin group and anti-Fas antibody group were significantly different from the inhibitor group (P<0.01). CONCLUSION: The Fas-dependent signalling is the important pathway of azurin inducing apoptosis in U2OS cells. The up-regulation of csepase-8 may play an important role.     

Induction of apoptosis in mouse fibroblast cell line L929 by arachidonic acid

AIM: To observe whether arachidonic acid (AA) could induce apoptosis in mouse fibroblast cell line L929 and the potential mechanism involved. METHODS: The viability and damaged degree of L929 was monitored by MTT and the release of lactate dehydrogenase (LDH). Lipid peroxidation in L929 was measured as malondialdehyde (MDA) content by colorimetric assay. Hoechst 33258 staining was used to observe AA-induced morphological changes. Agarose gel electrophoresis was used to detect DNA fragmentation. RESULTS: Treatment of L929 cell with AA for 24 h, in the range of 40-160 μmol/L, caused a great decrease in cell survival and increased MDA contents and the release of LDH simultaneously (P＜0.01). Cells treated by 160 μmol/L AA showed typical morphological changes of apoptosis. A "ladder" pattern representing fragmentation of DNA into oligonucleosome length fragments was observed 24 h after AA treatment. CONCLUSION: Higher concentration of arachidonic acid (80-160 μmol/L) induced apoptosis in mouse fibroblast cell line L929. The mechanism of its action might be related to lipid peroxidation.     

Effect of CoQ10 on apoptosis and proliferation of cultured mouse microvascular endothelial cells and it's mechanism

AIM: To study the inhibitory effect of CoQ₁₀ on the apoptosis of microvascular endothelial cells and it's probable mechanism. METHODS: Using serum pharmacology method and cytoflowmetery, the effects of CoQ₁₀ at different concentrations on apoptosis and proliferation in cultured mouse brain microvascular endothelial cells (bEnd.3) were investigated. The expression of Fas protein and Bcl-2 protein were observed with immunocytochemical method (ABC). RESULTS: The cell apoptosis was inhibited significantly in CoQ₁₀ groups (50 μL and 25 μL) in cultured bEnd.3 cells. The results of immunocytochemical staining showed that the expressions of Fas protein was inhibited and Bcl-2 protein was stimulated significantly in CoQ10 group with above concentration. But there was no significant change in cell proliferation. CONCLUSIONS: CoQ₁₀ may inhibit apoptosis of microvascular endothelial cells (bEnd.3) via up-regulation of Bcl-2 and down-regulation of Fas. Authors suggest that this is one of the protection mechanisms of CoQ₁₀ from dysfunction of microvascular endothelial cells.     

Effect of activated protein C on apoptosis in human umbilical vein endothelial cells induced by lipopolysaccharide

AIM:To study the effect of activated protein C (APC) at different concentrations on apoptosis of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS).METHODS:The HUVECs were induced by LPS (1.0 mg/L) as apoptotic model that was administered by different concentration of APC (10 μg/L or 50 μg/L). Meanwhile, the control group and induced apoptosis group induced by LPS (1.0 mg/L) stimulation were also set up. The changes of cellular ultrastructures were observed under electron microscope. The DNA ladder and TUNEL fluorescent staining were measured in cells. Annexin-Ⅴ/PI double staining was used to measure the cell apoptosis rate by flow cytometry. Cell survival rate was measured by MTT assay. The proliferating cell nuclear antigen (PCNA) expression levels in cells were also measured by Western blotting to reflect the proliferation of the cells.RESULTS:There were significant apoptotic changes in the cells induced by LPS, but the apoptotic changes were reduced and apoptosis rates were decreased in the cells treated with APC. Meanwhile, cell survival rate and the protein levels of PCNA were increased after APC treatment, particularly at the concentration of 50 μg/L, which showed difference when compared with those induced apoptosis group by LPS (P<0.05).CONCLUSION:APC can inhibit HUVECs apoptosis induced by LPS and promote cell proliferation, thus protect the cells from injury.     

Expression of apoptosis signal protein induced by Fas in labial salivary gland of patients with primary Sjgren’s syndrome

AIM:To study the expression of apoptosis signal proteins induced by Fas in labial salivary gland of patients with primary Sjgren’s syndrome (pSS), and investigate the possible pathway of the cell signaling transduction in pSS. METHODS:Biopsies of minor submucosal labial salivary gland were obtained from 32 patients with pSS and 8 normal controls. Fas, FasL and FADD were detected by Western blotting. Caspase-3 was measured by immunohistochemistry and CAD mRNA expression was determined by RT-PCR. RESULTS:The expressions of Fas, FasL, FADD, caspase-3 and CAD mRNA in labial salivary glands of patients with pSS were significantly higher than those in control group (P＜0.05). CONCLUSION:The apoptosis signalling transduction in labial salivary glands of patients with pSS may be that the product of FasL combined with Fas activates caspase-8 through FADD, then ICE family is cascaded, and finally CAD is splited by caspase-3 from ICAD, which catalyzes the degradation of DNA.      

Quercetin regulates Fas expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To investigate the role of 1, 4, 5- trisphosphate inositol (IP3) and Fas gene expression in apoptosis of HepG2 cells induced by quercetin. METHODS: HepG2 cells were treated with quercetin at different concentrations (including 20, 40, 60, 80 μmol/ L) for 72 h and treated with 60 μmol/ L quercetin for 6 h, 12 h, 24 h, 48 h and 72 h. IP3, Fas mRNA, Fas protein and apoptosis rate were assayed by IP3 - ［3H］ Birtrak assay, RT-PCR, Western blotting and flow cytometry, respectively. RESULTS: When HepG2 cells were incubated with different concentrations of quercetin for 72 h, the IP3 content was lower than those in control. Fas mRNA expression, Fas protein expression and the apoptosis rate were higher than those in control. When HepG2 cells were incubated with quercetin for 6 h, 12 h, 24 h, 48 h, 72 h, the IP3 contents were lower than those in control incubated with 60 μmol/L quercetin for 12 h. Fas mRNA expression was higher than that in control incubated with 60 μmol/L quercetin for 12 h . Fas protein expression was higher than that in control. The apoptosis rate was significantly higher than that in control incubated with 60 μmol/L quercetin for 24 h (P<0.01). CONCLUSION: Quercetin induces apoptosis of HepG2 cells by reducing IP3 production and upregulating Fas gene expression.     

Apoptosis induced by berbamine in K562 cells correlates with the expression levels of bcr/abl gene and P210

AIM: To explore the effect of berbamine(BER) on apoptosis in K562 cells and its possible molecular mechanisms. METHODS: The apoptosis rate was measured by flow cytometry while electron microscopy and DNA electrophoresis were used to evaluate the characteristic changes of apoptosis, RT-PCR and Western blot were used to examine the expression levels of apoptosis related gene bcr/abl and BCR/ABL protein. RESULTS: By FCM, the apoptosis rate of K562 cells treated with 8.0 mg/L BER for 24 h and 72 h increased from (29.20±3.82)% to (61.77±4.35)% (P＜0.01); The typical apoptosis morphologic changes and the DNA ladder were more clearly observed. After treated with BER 0 to 16.0 mg/L for 24 h, the expression levels of bcr/abl mRNA and P210 (semiquantity value) decreased quickly from 1.19±0.02 to 0.73±0.02 (P＜0.01) and from 1.04±0.02 to 0.63±0.01 (P＜0.01), respectively. CONCLUSION: BER induces apoptosis of K562 cells in a time-and-concentration-dependent manner, the decline of bcr/abl mRNA and P210 may play an important role in the apoptotic effect of BER in K562 cells. BER could be used as a new clinical trials for bcr-abl⁺ diseases such as CML.     

Effect of homocysteine on the apoptosis of cultured human umbilical vein endothelial cells

AIM: To investigate the effect of homocysteine(Hcy) on the apoptosis of endothelial cells (EC). METHODS: First-passaged human umbilical vein endothelial cells (hUVEC) were cultured with M199 containing 3 mmol/L Hcy. hUVEC apoptosis was detected as follow: demonstration of nuclear changes by Hoechst 33258 staining, agarose gel electrophoresis of DNA fragments, detection of apoptotic cells by flow cytometry following Annexin V-PI doubled stain, Western blot for P53 and Bax protein detection and colorimetry detecting caspase-3 activity. RESULTS: Compared with control, homocysteine induced characteristic apoptotic changes in hUVEC. The chromosomal DNA of hUVEC appeared "DNA ladder" by agarose gel electrophoresis. Apoptotic cells were increased significantly (P<0.01, n=3). Hcy promoted the expression of protein Bax, P53 (P<0.01, n=3) and enhanced the activity of caspase 3 (P<0.05, n=3). CONCLUSION: Homocysteine induces apoptosis in cultured hUVEC.     

Inhibitory effect of geinistein on apoptosis in hUVECs induced by MCP-1

AIM: To study the inhibitory effect of genistein on apoptosis in human umbilical vein endothelial cells (hUVECs) induced by monocyte chemotactic protein-1 (MCP-1). METHODS: The hUVECs were cultured in vitro and identified. Growth-arrested hUVECs were stimulated with genistein at different concentrations (0.1 μmol, 1.0 μmol, 10 μmol, 100 μmol) and co-treated with MCP-1 (10 μg/L). The survival rates of hUVECs were detected by MTT assay. The cell cycle and DNA content were detected by flow cytometry. To explore the possible mechanism of the genistein interventions, the expressions of Bcl-2, Fas and Bax proteins were detected by flow cytometry and Western blotting.RESULTS: Genistein increased the survival rate and the level of Bcl-2, inhibited Fas and Bax, decreased the ratios of apoptosis compared with MCP-1-induced hUVECs apoptotic group in a dose-dependent-manner. CONCLUSION: Genistein inhibits the apoptosis induced by MCP-1 and the inhibitory effect was relative to the dose of genistein. Its mechanism might be involved in the down-regulation of Fas and Bax expressions and the up-regulation of Bcl-2.     

STAT3 mediates PDGF-BB-induced Pim-1 expression in rat vascular smooth muscle cells

AIM: To study the expression of Pim-1 in vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor BB (PDGF-BB). METHODS: VSMCs isolated from rats were treated with different concentrations of PDGF-BB for different time. The proliferation of VSMCs was detected by cell counting. The mRNA expression of Pim-1 was measured by real-time RT-PCR. The STAT3 activity was determined by Western blotting. Actinomycin D, AG490, and small interfering RNA (siRNA) for Pim-1 or STAT3 were used to investigate the underlying mechanisms. RESULTS: Pim-1 gene silencing attenuated the proliferation of VSMCs in response to PDGF-BB. The mRNA expression of Pim-1 was up-regulated by PDGF-BB at concentrations of 10 μg/L~50 μg/L for 1 h, and was maximally induced at the concentration of 20 μg/L. The time of Pim-1 mRNA expression maximally occurred 30 min after PDGF-BB exposure. Incubation of VSMCs with PDGF-BB resulted in a significant activation of STAT3. VSMCs pretreated with actinomycin D showed a significant decrease in the mRNA expression of Pim-1. Treatment with AG490 or knockdown of STAT3 in VSMCs resulted in inactivation of STAT3, and significantly suppressed the mRNA expression of Pim-1. CONCLUSION: PDGF-BB-induced VSMC proliferation is partly attributed to Pim-1. VSMCs strongly increase Pim-1 mRNA upon stimulation with PDGF-BB, and STAT3 signaling pathway appears to be efficient for regulation of Pim-1 expression. This process may play a critical role in development of vascular remodeling.     

Tumor necrosis factor-related apoptosis-inducing ligand participates in injury of vascular endothelial cells induced by rapamycin in vitro

AIM: To investigate the effects of rapamycin on apoptosis, proliferation, migration ability and tumor related apoptosis inducing ligand(TRAIL) in cultured human umbilical vein endothelial cells(HUVECs). METHODS: Cultured HUVECs were treated with rapamycin at the concentrations of 0, 1, 10 and 100 μg/L for 24 h. The cell proliferation was measured by CCK-8 method. The cell migration ability was detected by Transwell chambers and wound healing test. The apoptotic index of HUVECs was quantitatively determined by measuring the activation of caspase-3. The morphological changes of the apoptotic cells were observed by DAPI staining. The expression of TRAIL was detected by Western blotting. RESULTS: A 24 h-incubation with rapamycin(1-100 μg/L) caused significant cell loss associated with the increase in apoptosis, as quantified by the determination of caspase-3 activity(P<0.01) in HUVECs. Obvious apoptotic morphology was observed by DAPI staining in HUVECs incubated with rapamycin. Rapamycin at the concentrations of 1-100 μg/L also impaired the migration ability of HUVECs(P<0.01). In addition, rapamycin(10-100 μg/L) inhibited the proliferation of HUVECs, whereas rapamycin at 1 μg/L had no such effect(P<0.01). Rapamycin(10-100 μg/L) also induced TRAIL expression in a dose-dependent manner(P<0.01). CONCLUSION: Rapamycin induces apoptosis, and inhibits the proliferation and migration of HUVECs. The up-regulation of TRAIL might be related to the injury of vascular endothelial cells caused by rapamycin.     

Inhibitory effects of a cyclooxygenase-2 inhibitor,NS398,on cancer cells

AIM:To study the mechanism of growth inhibitory effect of a selective cyclooxygenase-2 (COX-2) inhibitor,NS-398,on cancer cells.METHODS:The esophageal cancer cell line (EC9706),which expresses COX-2 constitutively,and hepatocellular carcinoma cell line (SMMC7721),which expresses no COX-2,were studied.The cell lines were incubated with NS-398 at doses of 10,20,50,100 μmol/L for 24 h,48 h and 72 h.Antiproliferation effect was measured by ［3H］-TdR incorporation.The cell apoptosis were determined by flow cytometry (FCM) and DNA fragmentation analysis.Survivin was detected by immunocytochemical technique.RESULTS:The growth inhibition was induced by NS398 in a dose- and time-dependent manners in both cell lines.FCM analysis revealed a high sub-G1 cell peak in EC9706 group and agarose electrophoresis showed marked apoptosis ladder pattern.However,no apoptosis was observed in SMMC7721 cells treated with NS-398.The difference of apoptosis percentage in EC9706 and SMMC7721 was (45.23±1.08)% and (3.05±0.15)% (P<0.01).After 24 h incubation with NS-398 at concentration of 100 μmol/L,the expression of survivin was markedly reduced in EC9706,no change was observed in SMMC7721.CONCLUSION:NS-398 suppresses cell growth in cancer cell lines by different mechanism.NS-398 suppresses cell growth and increases apoptosis in the cancer cells that expresses COX-2.     

Hypoxia promotes apoptosis of neural stem cells and down-regulates miR-26a

AIM: To investigate the effect of cobalt chloride (CoCl₂) on the apoptosis of neural stem cells (NSCs) and the expression of microRNA-26a (miR-26a) in vitro, and to explore the mechanisms of NSC apoptosis induced by CoCl₂. METHODS: NSCs were exposed to CoCl₂ at different doses (200~600 μmol/L) for 24 h. The cell viability and apoptosis were measured by CCK-8 assay and TUNEL method. The expression of miR-26a-3p, miR-26a-5p, GSK-3β, caspase-3, Bcl-2 and Bax was examined by real-time PCR. The protein levels of Bcl-2 and Bax were detected by Western blotting. RESULTS: The cell viability was inhibited and the apoptosis of NSCs was increased significantly by CoCl₂ in a dose-dependent manner (P<0.05). CoCl₂ at concentration of 400 μmol/L for 24 h was used to induce apoptosis and the expression of miR-26a was down-regulated compared with control (P<0.05). Exposure to CoCl₂ at concentration of 400 μmol/L up-regulated the expression of GSK-3β, caspase-3 and Bax, down-regulated the expression of Bcl-2 and Bcl-2/Bax (P<0.05). CONCLUSION: CoCl₂ at concentration of 400 μmol/L induces the apoptosis of NSCs obviously. CoCl₂ may induce the NSC apoptosis by mitochondrial apoptotic pathway. Declining miR-26a may be related to NSC apoptosis.     

Changes and significance of Fas and cFLIP in apoptosis of human rhabdomyosarcoma cells induced by combination of TRAIL and cisplatin

AIM: To investigate the synergistic induction of apoptosis in rhabdomyosarcoma cells by the combination of TRAIL or TRAIL gene with cisplatin. METHODS: Rhabdomyosarcoma cells were treated with TRAIL, Ad／GT-TRAIL, cisplatin, respectively or the combination for 3 days. The cytotoxicity was observed by MTT assay. The apoptotic rates and the expression rates of Fas protein were measured by flow cytometry (FCM). The expression of cFLIP mRNA was determined by RT-PCR. RESULTS: Rhabdomyosarcoma cells were treated with Ad／ GT-TRAIL and TRAIL (100.0 μg/L), the cytotoxicity index were 52.5% and 43.5%, the percentage of apoptotic cells were 12.95% and 10.26%, respectively. Combined with cisplatin, the cytotoxicity index and the percentage of apoptotic cells were increased significantly (P<0.05). The expression of Fas protein in rhabdomyosarcoma cells was up-regulated and the expression of cFLIP was down-regulated with cisplatin, which were paralleled by the apoptotic rates. CONCLUSION: Combinatiion of Ad／GT-TRAIL or TRAIL and cisplatin has synergistic apoptosis-inducing effects on rhabdomyosacoma cells.     

Apoptosis induced by simvastatin in rat vascular smooth muscle cells through calpain and caspase-3-dependent pathways

AIM:Hydroxymethylglutaryl CoA (HMG-CoA) reductase inhibitors, such as simvastatin, have been shown to reduce atherosclerotic cardiovascular morbidity and mortality by mechanisms unrelated to its lipid-lowering effect. Several studies have shown that simvastatin induces apoptosis in a varieties of cell lines including vascular smooth muscle cells (VSMC). The aim of this study was to investigate the signal pathways involved in apoptosis induced by simvastatin.METHODS:Cultured VSMC were treated with simvastatin. Calpain activity was determined by measuring Ca²⁺ ionophore-specific calpain substrate (suc-LLVY-AMC), caspase-3 activation was detected by Western blot, and apoptotic changes were distinguished by annexin Ⅴ binding and DNA laddering.RESULTS:After incubated with 30 μmol/L simvastatin for 8 h, calpain activity had a marked increase (P<0.05, n=4) and reached to more than 3-fold of control at 12 h (P＜0.01). Caspase-3 also activated by simvastatin after 12 h. PD150606, a cell-permeable selective calpain inhibitor, decreased simvastatin-induced apoptosis rate from 24.2%±1.7% to 9.5%±1.9% (P＜0.01) and also prevented simvastatin-induced DNA laddering. Furthermore, 100 μmol/L PD150606 efficiently inhibited simvastatin-induced caspase-3 activation.CONCLUSION:Simvastatin induces apoptosis by activating caspase-3 via calcium-dependent protease calpain.     

Apoptosis induced by 5-fluorocytosine in human pancreatic cancer cells genetically modified to express cytosine deaminase

AIM:To elucidate the pattern of 5-fluorocytosine(5-FC) induced apoptosis and its role in gene therapy for human pancreatic cancer. METHODS: The human pancreatic cancer SW1990 cells(CEA-producing) were infected with recombinant adenoviruses(Adex1CEA-prCD or Adex1CEA-prZ).Cytosine deaminase(CD) expression was examind by western blot. Apoptosis induced by 5-FC in human pancreatic cancer SW1990 cells genetically modified to express cytosine deaminase was investigated by applying electron microscopy, DNA electrophoresis and flow cytometry analysis techniques. RESULTS: The SW1990 cells infected with Adex1CEA-prCD were treated with 5-FC at 100 μmol·L^-1 for 48 h, cell apoptosis occurred. Typical apoptosis morphological feature appeared and DNA ladder could be demonstrated on DNA electrophoresis. Apoptosis peak was also showed by flow cytometry. Apoptotic cells accounted for 34.6% of the cell population. Cells in G₁, S and G₂/M phase of cell cycle were 64%, 11% and 7%, respectively. CONCLUSION: The apoptosis induced by 5-FC may be a primary mechanism in CD gene therapy for pancreatic cancer.