APOPTOSIS,REDIFFERENTIATION AND ARRESTING PROLIFERATION SIMULTANEOUSLY TRIGGERED BY OXIDATIVE STRESS IN HUMAN HEPATOMA CELLS

The effects of oxidative stress (ascorbic acid—ferrous system) on the proliferation, differentiation and apoptosis of the human hepatoma cell SMMC-7721 were studied. Oxidative stress significantly inhibited cell proliferation and induced morphological differentiation. Whatever the indices related with cell malignancy, such as α-fetoprotein and c-glutamyltranspeptidase or the index related with cell differentiation, such as tyrosine-α-ketoglutarate transaminase, all inclined evidently to normalization. The tumour's clonogenic potential decreased significantly. Moreover, together with differentiation, the phenomenon of apoptosis was found by the appearance of apoptotic bodies, detached cells, and apoptotic morphological feature. Although, their DNA was not degraded into oligonucleosomal fragmentation, the DNA was cut into larger fragments (about 21.2kbp) of a size associated with chromatin loops. These findings indicated that oxidative stress can induce both differentiation and apoptosis simultaneously in tumour cells. All the results showed that oxidative stress may initiate the tumour cells reverse transformation. The possible mechanism of the differentiation and apoptosis induced by oxidative stress may be related to the lipid peroxidation of cell membrane.     

Oxidative stress-mediated apoptosis. The anticancer effect of the sesquiterpene lactone parthenolide

The sesquiterpene lactone parthenolide, the principal active component in medicinal plants, has been used conventionally to treat migraines, inflammation, and tumors. However, the antitumor effects of parthenolide and the mechanism(s) involved are poorly understood. We found that parthenolide effectively inhibits hepatoma cell growth in a tumor cell-specific manner and triggers apoptosis of hepatoma cells. Parthenolide triggered apoptosis in invasive sarcomatoid hepatocellular carcinoma cells (SH-J1) as well as in other ordinary hepatoma cells at 5-10 microm concentrations and arrested the cell growth (at G(2)/M) at sublethal concentrations (1-3 microm). During parthenolide-induced apoptosis, depletion of glutathione, generation of reactive oxygen species, reduction of mitochondrial transmembrane potential, activation of caspases (caspases-7, -8, and -9), overexpression of GADD153 (an oxidative stress or anticancer agent inducible gene), and subsequent apoptotic cell death was observed. This induced apoptosis could be effectively inhibited or abrogated by an antioxidant N-acetyl-l-cysteine, whereas l-buthionine-(S,R)-sulfoximine enhanced it. Furthermore, stable overexpression of GADD153 sensitized the cells to apoptosis induced by parthenolide, and this susceptibility could be reversed by transfection with an antisense to GADD153. Parthenolide did not alter the expression of Bcl-2 or Bcl-X(L) proteins during apoptosis in hepatoma cells. Oxidative stress may contribute to parthenolide-induced apoptosis and to GADD153 overexpression in a glutathione-sensitive manner. The sensitivity of tumor cells to parthenolide appears to result from the low expression level of the multifunctional detoxification enzyme glutathione S-transferase pi. Finally, parthenolide and its derivatives may be useful chemotherapeutic agents to treat these invasive cancers.     

Genistein对大鼠垂体前叶细胞增殖的抑制作用

应用细胞培养、^3H-TdR掺入、流式细胞和电镜技术,观察酪氨酸蛋白激酶（PTK）抑制剂genistein对正常大鼠垂体前叶细胞和垂体瘤细胞株AtT-20增殖的影响,并探讨其可能的机制。结果显示：genistein作用48h后可明显抑制正常大鼠垂体前叶细胞和垂体瘤细胞株AtT-20增殖。流式细胞仪检测发现,50和100μmol/L genistein可将AtT-20细胞阻断于G0／G1期及G2／M期,并出现凋亡峰,凋亡率分别灰19.9％和36.4％。电镜照片显示有凋亡细胞。结果表明,PTK抑制剂可以明显抑制正常大鼠垂体前叶细胞和垂体瘤细胞株AtT-20的殖,并诱导细胞凋亡,说明PTK活性对细胞增殖和分化有重要作用。     

Oxidative stress modulates osteoblastic differentiation of vascular and bone cells

Oxidative stress may regulate cellular function in multiple pathological conditions, including atherosclerosis. One feature of the atherosclerotic plaque is calcium mineral deposition, which appears to result from the differentiation of vascular osteoblastic cells, calcifying vascular cells (CVC). To determine the role of oxidative stress in regulating the activity of CVC, we treated these cells with hydrogen peroxide (H(2)O(2)) or xanthine/xanthine oxidase (XXO) and assessed their effects on intracellular oxidative stress, differentiation, and mineralization. These agents increased intracellular oxidative stress as determined by 2,7 dichlorofluorescein fluorescence, and enhanced osteoblastic differentiation of vascular cells, based on alkaline phosphatase activity and mineralization. In contrast, H(2)O(2) and XXO resulted in inhibition of differentiation markers in bone osteoblastic cells, MC3T3-E1, and marrow stromal cells, M2-10B4, while increasing oxidative stress. In addition, minimally oxidized low-density lipoprotein (MM-LDL), previously shown to enhance vascular cell and inhibit bone cell differentiation, also increased intracellular oxidative stress in the three cell types. These effects of XXO and MM-LDL were counteracted by the antioxidants Trolox and pyrrolidinedithiocarbamate. These results suggest that oxidative stress modulates differentiation of vascular and bone cells oppositely, which may explain the parallel buildup and loss of calcification, seen in vascular calcification and osteoporosis, respectively.     

Oxidative stress and activation of proteasome protease during serum deprivation-induced apoptosis in rat hepatoma cells; inhibition of cell death by melatonin

Growth factor deprivation-induced apoptosis has been shown in various cell systems and is recognized as one of the standard models for the study of programmed cell death. The mechanism of induction of apoptosis by serum deprivation is still not clear. The objective of the present study was to investigate if serum-deprivation causes oxidative stress, which then leads to apoptotic death. We have demonstrated that indeed, there was a significant increase in reactive oxygen species following serum deprivation of 5123tc hepatoma cells. Furthermore, treatment with anti-oxidants; melatonin or vitamin E, prevented cell death caused by serum-deprivation. We also demonstrated that there was activation of proteasome proteases and decrease in glutathione levels following serum deprivation. Interestingly, melatonin treatment blocked these changes and rescued the cells from apoptosis induced by serum-deprivation. These results indicated that oxidative stress may play a causal role in the induction of apoptosis induced by serum deprivation.     

过氧化氢预处理对抗氧化应激诱导的PC12细胞凋亡

氧化应激可明显地诱导细胞凋亡。本研究旨在探讨H2O2预处理能否对H2O2诱导的PC12细胞凋亡生产保护作用及ATP敏感性K^ （ATP-sensitive potassinm,KATP）通道在其中的作用。采用PI染色流式细胞仪（flow cytometry, FCM）检测PC12细胞凋亡。结果表明,经10μmol/L H2O2预处理90min的PC12细胞,分别在20、30、50和100μmol/L H2O2作用24h后,其细胞凋亡率明显下降,与未经H2O2的预处理的PC12细胞相比,差异极显著（P＜0.01）,表明H2O2预处理对H2O2诱导PC12细胞凋亡具有保护作用。用10μmol/L的KATP通道激动齐pinacidil(Pin)可显著减少30和50μmol/L H2O2诱导的PC12细胞凋亡,10μmol/L的KATP通道拮抗齐glybenclamide（Gly）则可显著地抑制甚至取消KATP通道激动剂Pin对H2O3诱导PC12细胞凋亡的保护作用,但并不影响H2O2预处理对H2O2诱导PC12细胞凋亡的保护作用;然而,当联合应用H2O2预处理与Pin时,对PC12细胞凋亡的保护作用显大于各自的细胞凋亡作用。提示KATP通道开放不仅对H2O2诱导PC12细胞凋亡具有保护作用,而且与H2O2预处理一起产生抗PC12细胞凋亡的协同作用。但KATP通道开放可能不参与H2O2预处理的适应性保护作用。     

Osteogenic oxysterols inhibit the adverse effects of oxidative stress on osteogenic differentiation of marrow stromal cells

The osteoporosis that occurs with aging is associated with reduced number and activity of osteoblastic cells. Aging, menopause, and osteoporosis are correlated with increased oxidative stress and reduced antioxidant defense mechanisms. We previously demonstrated that oxidative stress induced by a variety of compounds such as xanthine/xanthine oxidase (XXO) and minimally oxidized LDL (MM-LDL) inhibit the osteogenic differentiation of osteoprogenitor cells. Oxysterols are a family of products derived from cholesterol oxidation that have important biological activities. Recently, we reported that a specific oxysterol combination consisting of 22(S)- or 22(R)-hydroxycholesterol and 20(S)-hydroxycholesterol has potent osteogenic properties in vitro when applied to osteoprogenitor cells including M2-10B4 (M2) marrow stromal cells. We now demonstrate that this osteogenic combination of oxysterols prevents the adverse effects of oxidative stress on differentiation of M2 cells into mature osteoblastic cells. XXO and MM-LDL inhibited the osteogenic differentiation of M2 cells, demonstrated by the inhibition of markers of osteogenic differentiation: alkaline phosphatase activity, osteocalcin expression and mineralization. Treatment of M2 cells with osteogenic oxysterol combination 22(S)- and 20(S)-hydroxycholesterol both blocked and reversed the inhibition of osteogenic differentiation produced by XXO and MM-LDL in these cells. The protective effect of the oxysterols against oxidative stress was dependent on cyclooxygenase 1 and was associated with the osteogenic property of the oxysterols. These findings further demonstrate the ability of the osteogenic oxysterols to positively regulate osteogenic differentiation of cells, and suggests that the use of these compounds may be a novel strategy to prevent the adverse effects of oxidative stress on osteogenesis.     

过氧化氢预处理对H9c2心肌细胞氧化应激损伤的保护作用

目的：活性氧介导的氧化损伤是缺血再灌注损伤的重要机制,本研究通过观察H2O2预处理对氧化损伤的H9c2心肌细胞存活率和细胞凋亡的影响,探讨其保护H9c2心肌细胞的作用机制。方法：体外培养H9c2心肌细胞,取对数生长期细胞用于实验研究。建立H2O2预处理抵抗高浓度H：O：诱导的细胞氧化损伤模型,实验分组如下：（1）正常对照组（CTL）;（2）损伤组（INJURY）;（3）预处理组十损伤组（PC）。应用CCK8法检测细胞存活率;试剂盒检测胞内MDA水平和T．sOD活性;Hoechst33258染色观察凋亡形态;Annexin-V／PI双染与流式细胞术检测细胞凋亡率。结果：25vLmol／L的H202预处理90rain能明显地保护H9c2心肌细胞抵抗400μmol／LH2O2诱导的氧化损伤,提高细胞存活率,下调MDA水平,上调SOD活性,抑制细胞凋亡,降低细胞凋亡率。结论：低浓度H2O2预处理能减轻H9c2心肌细胞的氧化损伤,抑制氧化损伤诱导的心肌细胞凋亡,具有很好的抗氧化损伤和抗心肌细胞凋亡的保护作用,其作用机制可能与细胞SOD活性上调有关。H2O2预处理为临床治疗心肌缺血／再灌注损伤提供了一项新策略。     

The prevention of the staurosporine-induced apoptosis by Bcl-XL, but not by Bcl-2 or caspase inhibitors, allows the extensive differentiation of human neuroblastoma cells

Staurosporine is one of the best apoptotic inducers in different cell types including neuroblastomas. In this study we have compared the efficiency and final outcome of three different anti-apoptotic strategies in staurosporine-treated SH-SY5Y human neuroblastoma cells. At staurosporine concentrations up to 500 nm, z-VAD.fmk a broad-spectrum, noncompetitive inhibitor of caspases, reduced apoptosis in SH-SY5Y cells. At higher concentrations, z-VAD.fmk continued to inhibit caspases and the apoptotic phenotype but not cell death which seems to result from oxidative damage. Stable over-expression of Bcl-2 in SH-SY5Y protected cells from death at doses of staurosporine up to 1 microm. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated and cells died by apoptosis. Therefore, we conclude that Bcl-2 increased the threshold for apoptotic cell death commitment. Over-expression of Bcl-X(L) was far more effective than Bcl-2. Bcl-X(L) transfected cells showed a remarkable resistance staurosporine-induced cytochrome c release and associated apoptotic changes and survived for up to 15 days in 1 microm staurosporine. In these conditions, SH-SY5Y displayed a remarkable phenotype of neuronal differentiation as assessed by neurite outgrowth and expression of neurofilament, Tau and MAP-2 neuronal specific proteins.     

Radiosensitization mechanism of riboflavin in vitro

Riboflavin, suggested to be a radiosensitizer, was studied in murine thymocytes and human hepatoma L02 cell line in vitro with MTT method and fluorescence microscopy. When the murine thymocytes treated with 5-400 μmol/L riboflavin were irradiated by 5 Gy 60Co γionizing radiation, the low concentration groups, i.e. treated with 5-50 μmol/L riboflavin, showed a different surviving fractions-time relating correlation compared with the high concentration groups, i.e. treated with 100-400 μmol/L riboflavin. The former had a high survival level at the end of irradiation, but which, after 4-h incubation, decreased rapidly to a low level. On the contrary, the high concentration groups showed a low survival level at the end of irradiation, and a poor correlation was found between the surviving fraction and the incubation time, after 4 h a little difference was observed. The results of fluorescence microscopy indicated that under low concentration conditions, the riboflavin localized mainly in nucleus (both pe