艾迪注射液对人肝癌细胞的增殖抑制作用和凋亡诱导活性

目的观察艾迪注射液对肝癌细胞株Hep3B和LM3的体外抗肿瘤作用。方法通过细胞的活力测定(MTS)法测定不同浓度艾迪注射液对Hep3B和LM3肝癌细胞的增殖抑制作用,流式细胞术分析不同浓度艾迪注射液对Hep3B和LM3肝癌细胞的诱导凋亡活性。结果艾迪注射液可显著抑制Hep3B和LM3肝癌细胞的增殖,并可有效诱导Hep3B和LM3肝癌细胞凋亡。结论艾迪注射液可有效抑制肝癌细胞增殖并诱导肝癌细胞凋亡,可能是艾迪注射液治疗肝癌发挥抗肿瘤疗效的重要机制。

An agonistic monoclonal antibody against DR5 induces ROS production, sustained JNK activation and Endo G release in Jurkat leukemia cells

We have previously reported that AD5-10, a novel agonistic monoclonal antibody against DRS, possessed a strong cytotoxic activity in various tumor cells, via induction of caspase-dependent and -independent signaling pathways. The present study further demonstrates that reactive oxygen species （ROS） were generated in abundance in Jurkat leukemia cells upon AD5-10 stimulation and that ROS accumulation subsequently evoked sustained activation of c-Jun N-terminal kinase （JNK）, loss of mitochondrial membrane potential, and release of endonuclease G （Endo G） from mitochondria into the cytosol. The reducing agent, N-acetylcysteine （NAC）, effectively inhibited the sustained activation of JNK, release of Endo G, and cell death in Jurkat cells treated by AD5-10. Moreover, a dominant-negative form of JNK （but not of p38） enhanced NF-κB activation, suppressed caspase-8 recruitment in death-inducing signaling complexes （DISCs）, and reduced adverse effects on mitochondria, thereby inhibiting AD5-10-induced cell death in Jnrkat leukemia cells. These data provide novel information on the DR5-mediated ceil death-signaling path- way and may shed new light on effective strategies for leukemia and solid tumor therapies.

Octreotide induces caspase activation and apoptosis in human hepatoma HepG2 cells

AIM: To investigate the role of octreotide on cellular proliferation and apoptosis of human hepatoma (HepG2) cells. METHODS: We studied cellular proliferation, apoptosis and the possible internal caspase-mediated apoptosis pathway involved, after treatment of HepG2 carcinoma cells with octreotide in comparison with the apoptosis caused by tumor necrosis factor-α (TNF-α). Activities of caspase-3, caspase-9, caspase-8 and caspase-2 were studied, while apoptosis was investigated through detection of DNA fragmentation and through identification of apoptotic cells with the annexin-V/propidium iodide flow cytometric method. RESULTS: After an initial increase in HepG2 cellular proliferation, a significant inhibition was observed with 10-8 mol/L octreotide, while TNF-α dose-dependently decreased proliferation. Early and late apoptosis was significantly increased with both substances. Octreotide significantly increased caspase-3, caspase-8 and caspase-2 activity. TNF-α signifi cantly increased only caspase-2. Cellular proliferation was decreased after treatment with octreotide or TNF-α alone but, in contrast to TNF-α, octreotide decreased proliferation only at concentrations of 10-8 mol/L, while lower concentrations increased proliferation. CONCLUSION: Our findings are suggestive of caspasemediated signaling pathways of octreotide antitumor activity in HepG2 cells, and indicate that measurements of serum octreotide levels may be important, at least in clinical trials, to verify optimal therapeutic drug concentrations.

Cobra phospholipase A2 protect cerebellar granule neurons from apoptosis with mechanism independent of enzymatic activity

A fraction of cobra （Naja naja atra） venom has been discovered to have protective effect on rat cultural cerebellar granule neurons （CGNs） from apoptosis induced by removing serum and reducing the extracellular potassium concentration from 25 to 5 mM. This component has been purified and identified as secreted phospholipase A2 （cobra sPLA2）. In order to study the relationship between the protection on CGNs and enzymatic activity of phospholipase A2, CGNs stained by Hoechst 33258 were quantified to determine survival rate under the fluorescence microscopy; the protective potencies on apoptosis of cultural CGNs were compared among cobra sPLA2, the cobra sPLA2 modified in carboxylate groups with water soluble carbodiimide and semicarbazide, and the heated cobra sPLA2 at 80℃ for 30 rain. The results showed that the CGN survival rate was unaffected significantly both in modified cobra sPLAz whose enzymatic activity of PLA2 had decreased by 80%, and in cobra sPLA2 adding 7, 7-Dimethyleicosadienoic acid, an inhibitor of sPLA2 at concentration of 10-fold IC50; contrary, the neuronal survival rate fell about 60% in heated cobra sPLA2, although its PLA2 activity only decreased by 10%. The protection on CGNs were also found in some of sPLA2s derived from venoms of bee, Naja naja mossambica, Crotalus atroxalso and Vipera Ammodytes Ammodytes but could not be found in other sPLA2s from bovine pancreas and Streptomyces violaceoruber. Above results suggest that the protection on CGNs of cobra sPLA2 is independent of its enzymatic activity.

N-acetylcysteine blocked hypoxia-reoxygenation induced apoptosis through ROS-p38 MAPK signaling pathway in neonatal rat cardiomyocytes

Objective Previous investigations have shown that N-acetylcysteine （NAC） could regulate diverse cell type＇s apoptosis. The purpose of this study was to evaluate the mechanism of NAC reversed apoptosis of cardiomyocytes induced by hypoxia-reoxygenation （H/R）. Methods Cardiomyocytes were treated with hypoxia 6 h and reoxygenation 72 h in the absence and presence of NAC （100/2mol/ L）. The ROS was assayed by using Image-iTTM LIVE green reactive oxygen species detection kit. The viability of cell was assayed with trypan blue. Early stages ofapoptosis were assessed by flow cytometry using Annexin V, and late stages of apoptosis were assessed using TUNEL system. Bcl2 and bax mRNA levels were determined by real-time quantitative PCR. Bcl2, bax, p38 and pp38 protein levels were determined by western blot. Results We found that H/R could markedly increase ROS generation and induce the apoptosis of cardiomyocytes （P〈0.01）. NAC （10012 mol/L） significantly reduced the generation of ROS and apoptosis （P all 〈0.01）. NAC also significantly reduced the protein ratio of pp38 and p38 and increased the RNA and protein ratio of bcl2 and bax （P all 〈0.01）. Conclusion The results showed that NAC significantly reduced apoptosis through inhibiting the phosphorylation of p38 signal pathway, which has potential value for clinical cardiac diseases （J Geriatr Cardio12009; 6：168-172）.