Role of thiocyanate, bromide and hypobromous acid in hydrogen peroxide-induced apoptosis

We have previously reported that H₂O₂-induced apoptosis in HL-60 human leukemia cells takes place in the presence of chloride, requires myeloperoxidase (MPO), and occurs through oxidative reactions involving hypochlorous acid and chloramines. We now report that when chloride is replaced by the pseudohalide thiocyanate, there is little or no H₂O₂-induced apoptosis. Furthermore, thiocyanate inhibits H₂O₂-induced apoptosis when chloride is present at physiological concentrations, and this occurs at thiocyanate concentrations that are present in human serum and saliva. In contrast, bromide can substitute for chloride in H₂O₂-induced apoptosis, but results in a lower percent of the cells induced into apoptosis. Hypobromous acid is likely a short-lived intermediate in this H₂O₂/MPO/bromide apoptosis, and reagent hypobromous acid and bromamines induce apoptosis in HL-60 cells. We conclude that the physiologic concentrations of thiocyanate found in human plasma could modulate the cytototoxicity of H₂O₂ and its resulting highly toxic MPO-generated hypochlorous acid by competing with chloride for MPO. Furthermore, the oxidative products of the reaction of thiocyanate with MPO are relatively innocuous for human leukemic cells in culture. In contrast, bromide can support H₂O₂/MPO/halide apoptosis, but is less potent than chloride and it has no effect in the presence of physiological levels of chloride.     

硒对铅暴露致神经损伤的拮抗作用研究

目的:研究铅暴露诱导的神经毒性损伤作用,明确铅暴露引发神经毒性损伤的部分机制以及硒的保护作用。方法:通过哺乳期染铅及补充硒建立铅暴露动物模型;通过TUNEL实验确定铅暴露引发的神经损伤;通过Western blot实验检测Bax、Bcl-2、Caspase-3水平确定铅暴露对凋亡途径的启动;并确证补硒在铅神经毒性作用下对机体的保护作用。结果:1.哺乳期铅暴露能够引起仔鼠海马神经细胞凋亡的发生;2.铅暴露能够诱导Bax/Bcl-2水平改变,激活Caspase-3。3.同时给予硒干预后,机体抗铅神经毒性能力显著增加。结论:1.铅暴露能够诱导海马部位神经毒性损伤,损伤可能通过启动凋亡途径而发生,2.补硒能够通过拮抗凋亡发生从而拮抗铅的神经毒性,产生保护作用。     

17-AAG对大鼠颈总动脉球囊损伤后内膜增生的影响及其作用机制

目的:研究17-丙烯胺-17去甲氧格尔德霉素(17-Allylamino-17-emethoxy-geldanamycin, 17-AAG)对球囊损伤后大鼠颈总动脉内膜增生的影响及可能作用机制。方法:将清洁级雄性SD大鼠36只按照随机数字法分为假手术组(Sham组)12只、球囊损伤组(Balloon injury, BI组)12只及17-AAG治疗组(17-AAG组)12只。采用2F Fogarty球囊建立大鼠颈总动脉球囊损伤组模型,17-AAG治疗组大鼠在建模后腹腔注射17-AGG(20 mg/kg 2d)。各组大鼠于球囊损伤3周后取损伤段颈总动脉,通过HE染色观察血管内膜形态学改变并评估内膜增生情况,免疫组化染色(Immunohistochemical staining,IHS)法检测血管壁增殖细胞核抗原(Proliferating cell nuclear antigen,PCNA)的表达,评估血管平滑肌细胞的增殖情况。流式细胞术检测血管平滑肌细胞的凋亡情况。结果:BI组、17-AAG组大鼠球囊损伤后颈总动脉内膜出现不同程度增生,内膜/中膜面积比(Intima area/Membrane area,I/M)均较Sham组显著升高(P0.05);17-AAG组的I/M较BI组明显下降(P0.05)。BI组、17-AAG组颈总动脉PCNA表达水平较Sham组明显升高(P0.05),较BI组显著降低(P0.05)。BI组、17-AAG组大鼠血管平滑肌细胞凋亡率较Sham组显著升高(P0.05);17-AAG组大鼠血管平滑肌细胞凋亡程度较BI组明显升高(P0.05)。结论:17-AAG对球囊损伤后颈总动脉内膜增生存在抑制作用,其机制可能是通过提高血管平滑肌细胞凋亡率影响其增殖程度。     

Synthesis and biological evaluation of new bisindole-imidazopyridine hybrids as apoptosis inducers

A series of diindolylmethanes (5a-t) were designed, synthesized, and examined for their cytotoxicity against four human cancer cell lines like prostate (DU-145), lung (A549), breast (MCF-7) and cervical cancer (HeLa). These results revealed that among all the hybrids, two (5k and 5r) were identified and exhibited significant cytotoxic effect against A549 cancer cells with IC₅₀ values of 1.65 ± 0.3 and 1.80 ± 0.8 µM respectively. To investigate the reasons for the cytotoxic activity, the conventional biological assays were carried out with 5k and 5r on the A549 cancer cells. Both hybrids led to the arrest of A549 cell lines at the G2/M phase of the cell cycle and strongly induced apoptosis. Further the apoptotic effects of 5k and 5r were confirmed by ROS, annexin-V FITC, and mitochondrial membrane potential. Moreover, structure–activity relationships were elucidated with various substitutions on these hybrids.     

miR-155对人椎间盘退变髓核细胞凋亡的作用及机制研究

目的:探讨mi R-155对人椎间盘退变髓核细胞凋亡的影响及其作用机制。方法:首先构建慢病毒表达载体,在293T细胞中获得重组慢病毒,然后感染椎间盘退变髓核细胞得到稳定过表达细胞系,同时设置空载体和空白细胞组对照。用荧光显微镜观察慢病毒载体的标签蛋白GFP的表达,分别提取三组细胞总RNA,采用RT-q PCR方法检测mi R-155的表达;通过流式细胞术检测细胞凋亡,Western-Blot检测细胞中凋亡相关蛋白FADD、Caspase-3、Bcl-2及Bax的表达,JC-1试剂盒检测细胞线粒体膜电位的变化情况。结果:在荧光显微镜下,经慢病毒感染的过表达细胞系和空载体细胞系均出现绿色荧光,而空白细胞组未见绿色荧光;RT-qPCR结果显示构建的稳定过表达细胞系(GV369-miR-155-NP)中mi R-155的表达水平较高,且与空载体细胞系及空白细胞对照组均呈显著性差异(P0.05);与空载体组(GV369-NP)及空白细胞对照组相比,过表达组(GV369-miR-155-NP)细胞凋亡率显著降低(P0.05),FADD、Caspase-3、Bax的表达水平均明显下降,而Bcl-2表达水平显著增加(P0.05)。结论:mi R-155可能通过靶向结合Caspase-3和FADD阻止FasL-Fas途径或通过线粒体途径抑制人椎间盘退变髓核细胞凋亡。     

Caveolin-1 sensitizes vascular smooth muscle cells to mildly oxidized LDL-induced apoptosis

Oxidized low-density lipoprotein (oxLDL)-induced apoptosis of vascular cells may participate to plaque instability and rupture. Caveolin-1 has emerged as an important regulator of several signal transduction pathways and processes that play a role in atherosclerosis. In this study we examined the potential role of caveolin-1 in the regulation of oxLDL-induced Ca²⁺ signaling and apoptosis in vascular smooth muscle cells (VSMC). Cells expressing caveolin-1 were more susceptible to oxLDL-induced apoptosis, and this was correlated with enhanced Ca²⁺ entry and pro-apoptotic events. Moreover, caveolin-1 silencing by small interfering RNA decreased the level of apoptotic cells after oxLDL treatment. These findings provide new insights about the potential role of caveolin-1 in the regulation of oxLDL-induced apoptosis in vascular cells and its contribution to the instability of the plaque.     

Assessment on pollution of Ochratoxin A in grain in China and its apoptosis effect on vitro-cultured human tubular kidney cells

Ochratoxin A (OTA) is nephrotoxic, immunosuppressive, and teratogenic in many species and is a possible human carcinogen. In this study, we investigated the OTA pollution situations of grains in northern China and the signaling pathway that mediated OTA-induced apoptosis in human tubular kidney cells (HKCs). Samples of grains collected from three representative areas were determined by using high-performance liquid chromatography fluorescence method. The effects of OTA on cell apoptosis, caspase-3, Bax, and Bcl-2 expression, and phosphorylation of c-Jun NH(2) terminal kinase (JNK) were detected in cultured HKCs via flow cytometry (FCM), Hoechst 33258 staining, and Western blot. It showed that OTA pollution of edible grains was very common in north China. OTA could affect caspase-3, Bax, and Bcl-2 expression and increased cell apoptosis in cultured HKCs. The JNK signalling pathway might play an important role during these cellular events.     

Bid truncation mediated by caspases-3 and -9 in vinorelbine-induced apoptosis

Vinorelbine is a chemotherapeutic vinca alkaloid clinically prescribed for non-small cell lung cancer and breast cancer. Here we studied the mechanism for vinorelbine-induced apoptosis in a human T-cell lymphoma. Although vinorelbine induces DNA fragmentation that is inhibited by specific peptide inhibitors for caspases-9 and -3 in Jurkat cells, caspase-8 deficiency retards vinorelbine-induced apoptosis. Activation of caspase-8 is also observed in vinorelbine-treated cells, and the activity is diminished when the caspase-3 activity is blocked by a specific peptide inhibitor, Ac-DNLC-CHO. Blocking of the Fas receptor with an antagonistic anti-Fas antibody does not affect vinorelbine-induced DNA fragmentation. These results suggest that vinorelbine-induced apoptosis is enhanced by the activation of caspase-8 via caspase-9-mediated activation of caspase-3, but not through a Fas-triggered signal. Western blotting suggests that vinorelbine cleaves caspase-3, -9 and -8 and reduces the amount of mitochondrial cytochrome c. Caspase-8 deficiency suppresses all of these events. A downstream substrate for caspase-8, Bid, is also cleaved in vinorelbine-treated cells, but the Bid truncation is also observed in caspase-8-deficient Jurkat cells. Importantly, recombinant caspases-3 and -9, as well as caspase-8, directly cleaves recombinant Bid in vitro. These results suggest that caspases-3 and -9 participate in Bid truncation, indicating a new mechanism for vinorelbine-induces apoptosis.