细胞质在顺铂诱导HL—60细胞凋亡中的作用

在建立Bcl-2高表达细胞及其对照细胞株基础上,用无细胞体系研究了细胞质在顺铂诱导HL-60细胞凋亡中的调控作用。实验结果显示,凋亡细胞胞质提取物可引起提取的正常细胞核的固缩及染色质边缘优,类似于完整的细胞凋亡的改变。Bcl-2高表达细胞的胞质提取物具有抗凋亡能力。上述结果说明引起凋亡的物质及对抗凋亡的物质在细胞质中都存在,说明细胞质在顺铂诱导的HL-60细胞凋亡中起着较重要的作用。     

顺铂诱导直肠癌HCT116细胞凋亡及其作用机制的研究

目的 研究顺铂诱导直肠癌HCT116细胞凋亡及其作用机制.方法 采用酶联免疫吸附法M30-ApoptosisTM-ELISA-kits、流式细胞仪测定不同浓度顺铂作用不同时间HCT116细胞的凋亡情况;West-ern-Blotting测定p53、p21、Bcl-2蛋白水平的表达.结果 顺铂可抑制直肠癌HCT116细胞生长,并呈时效(F=1129.383,P=0.000)和量效(F=125.267,P=0.000)关系;2.5、5.0、10.0、15.0 μmol/L顺铂分别作用直肠癌HCT116细胞0、12、24、48、72 h,通过亚G1峰检测其凋亡率,顺铂作用24、48、72 h细胞凋亡率与对照组比较差异有统计学意义(χ2值分别为5.669、14.110、12.221,P值分别为0.010、0.003、0.000),不同时间顺铂抑制HCT116细胞生长率的差异有统计学意义(χ2=14.008,P=0.003);不同时间顺铂诱导HCT116细胞释放CK18-Asp237-Asp396含量的比较,不同药物浓度结果差异有统计学意义(F=48.667,P=0.000),同时间结果差异有统计学意义(F=1194.394,P=0.000),Western-Blotting结果提示顺铂作用直肠癌HCT116细胞后p53、p21蛋白水平表达随着12、24、48、72 h与0 h比较逐步升高,p53蛋白(t值分别为9.873、-2.906、7.229、2.776,P值分别为0.000、0.007、0.000、0.011);p21蛋白(t值分别为-10.692、-8.867、-15.063、-16.281,P值分别为0.000、0.001、0.000、0.000);Bcl-2蛋白的表达无变化(t值分别为1.429、2.011、2.247、2.001,P值分别为0.178、0.069、0.053、0.062).结论 顺铂通过恢复pS3的功能,诱导肿瘤细胞凋亡,起到抑制肿瘤生长的作用.     

顺铂诱导直肠癌HCT116细胞凋亡及其作用机制的研究

Objective To study the apoptosis mediated by cisplatin on human colorectal tumors (HCT116) cell line and its mechanisms in vitro. Methods The apoptosis levels of HCT116 cells mediated by cisplatin at vari-ous time and in different concentration were measured by M30-ApoptosisTM -ELISA-kits and flow cytometry assay. The expressions of the protein p53,p21 and Bcl-2 were assessed through Western-Blotting. Results Cisplatin in-hibited the proliferation in a time-and dose -dependant manner ( F = 1129. 383, P = 0. 000 and F = 125. 267, P = 0. 000, respectively). The sub-G1 peak detected by flow cytometry at 24,48,72 hours of the apoptosis rates showed a significant difference between the experimental group and the control group (χ2= 5. 669,14.110,12. 221, P = 0. 010,0.003,0. 000,respectively). We found significant differences on the HCT116 cell growth between different time under cisplatin effect (χ2 = 14.008 ,P =0. 003 ). There were significant difference on the CK18-Asp237-Asp396 re-leased by HCT116 cell between different casplatin concentration (F =48. 667 ,P =0.000) as well as different times ( F = 1194. 394, P = 0.000). The Western-Blotting results indicated that the expression level of the p53 ( t = 9.873, -2.906,7. 229,2.776,P =0.000,0. 007,0. 000,0. 011 ) and p21 (t = - 10. 692, - 8. 867, - 15. 063, - 16.281, P = 0. 000,0.001,0.000,0.000 ,respectively) increased gradually, while there are no effect on the expression of the protein Bcl-2(t=1.429,2.011,2.247,2.001,P=0. 178,0.069,0.053,0.062,respectively). Conclusions Cis-platin can induce the apoptosis on HCT116 cells and thus inhibit the reproduction of tumor cells by recovering the function of p.53.     

重楼皂苷Ⅶ联合顺铂通过内质网应激诱导卵巢癌细胞凋亡

目的探讨重楼皂苷Ⅶ联合顺铂诱导卵巢癌SKOV3细胞凋亡的分子机制。方法从中草药重楼块根中分离提取重楼皂苷Ⅶ,然后联合顺铂处理SKOV3细胞,分为以下4组:0.1μg/ml重楼皂苷Ⅶ+1μg/ml顺铂组、1μg/ml重楼皂苷Ⅶ+1μg/ml顺铂组、10μg/ml重楼皂苷Ⅶ+1μg/ml顺铂组、对照组。培养24h和48h后,MTT法检测细胞增殖率,Hoechst染色检测细胞凋亡率,Real-time PCR检测凋亡相关基因caspase3以及内质网应激相关基因XBP1和ATF4的表达。结果重楼皂苷Ⅶ联合顺铂能有效促进SKOV3细胞凋亡,其凋亡是通过内质网应激激活caspase途径引起的。结论重楼皂苷Ⅶ能有效抑制肿瘤细胞生长,为临床上治疗卵巢癌提供了实验依据。     

DIM联合顺铂诱导PC-3细胞凋亡机制的研究

目的探讨顺铂(DDP)和3,3-二吲哚基甲烷(3,3-diindolylmethane,DIM)联合诱导人前列腺癌PC-3细胞凋亡的机制。方法采用免疫组化技术观察细胞内bcl-2,caspase3和caspase9蛋白的表达,利用RT-PCR和western blot检测bcl-2,caspase3和caspase9基因和蛋白表达变化。结果细胞培养及免疫组化可见:各给药组与对照组比较均有不同程度抑制PC-3细胞增殖并诱导其凋亡作用;RT-PCR结果显示:各给药组均能使caspase3和caspase9基因表达上调,bcl-2表达下调;western blot结果表明:各给药组与对照组相比caspase3和caspase9蛋白表达上调,bcl-2表达下调,且0.4mg.L-1DDP 60μmol.L-1DIM联合应用与10倍剂量DDP(4 mgL-1)单独应用效果相同。结论DIM与DDP均可抑制PC-3细胞增殖并诱导其凋亡;其细胞凋亡机制可能与上调caspase3、caspase9基因表达,下调bcl-2表达有关;DIM与DDP联合抗瘤具有明显的减毒增效作用。     

活性氧在荆花牡荆素诱导人肺癌A549细胞凋亡中的作用

目的 探讨紫花牡荆素(CAS)诱导人肺腺癌A549细胞凋亡及其机制.方法 体外培养A549细胞.MTT法测定CAS对A549细胞增殖的抑制;Annexin V/PI双染色分析细胞凋亡率;H2DCFH-DA探针流式细胞术分析活性氧(ROS)生成.结果 CAS能抑制人肺癌A549细胞增殖,呈浓度依赖性.Annexin V/PI法检测结果显示10 μmol/L的CAS作用A549细胞12 h、24 h、48 h后,其凋亡率分别为22.39%、38.66%、64.82%.H2DCFH-DA探针流式细胞术分析表明,完全培养基组、溶媒(0.1% DMSO)组对A459细胞作用0 h;CAS(10 μmol/L)对A549细胞作用3 h、6 h、12 h;N-乙酰-L-半胱氨酸(NAC,10 mmol/L)+CAS(10 μmol/L)组对A549细胞作用6 h的ROS生成水平分别为8.47、15.26、66.2、74.1、82.2、67.3,随着CAS作用时间延长,细胞内ROS水平增加.NAC对细胞凋亡有抑制作用.结论 CAS可诱导A549细胞凋亡,其作用机制可能与提高细胞内ROS产生增加有关.     

雷帕霉素联合顺铂诱导肝癌细胞凋亡和自噬及对细胞侵袭力的影响

目的研究雷帕霉素联合顺铂诱导肝癌细胞凋亡、自噬及其对细胞侵袭转移能力的影响。方法将雷帕霉素以及顺铂分别或联合作用于HepG-2细胞,采用MTT法检测HepG-2细胞增殖抑制情况,采用透射电镜、流式细胞仪检测HepG-2细胞凋亡,采用MDC染色、转染pGFP-LC3检测细胞自噬,用Transwell小室测定HepG-2细胞对细胞外基质的侵袭力。结果与对照组相比,单独应用雷帕霉素组未出现明显的细胞凋亡情况;单独应用顺铂组细胞凋亡情况较明显,凋亡率达到(21.27±3.65)%;顺铂联合应用雷帕霉素组细胞凋亡率最高,达到(43.33±5.64)%。雷帕霉素组、顺铂组和顺铂联合应用雷帕霉素组HepG-2细胞均出现点状的自噬泡,顺铂联合应用雷帕霉素组自噬泡数量显著高于雷帕霉素组和顺铂组。雷帕霉素组、顺铂组和顺铂联合应用雷帕霉素组对HepG-2细胞侵袭抑制率分别为(19.2±5.1)%、(47.8±4.1)%和(78.0±2.4)%。结论顺铂可直接诱导HepG-2细胞凋亡,雷帕霉素本身不诱导HepG-2细胞凋亡,但其可显著促进顺铂诱导的HepG-2细胞凋亡;同时顺铂和雷帕霉素均可直接诱导HepG-2细胞自噬,但两者联合应用促进HepG-2细胞自噬情况非常明显。体外侵袭实验结果表明顺铂联合应用雷帕霉素可显著降低HepG-2细胞的体外侵袭能力。     

顺铂诱导人骨肉瘤细胞凋亡及其分子机制的初步研究

目的　研究顺铂 (c DDP)对人骨肉瘤细胞的生物学效应及其作用机制。方法　骨肉瘤细胞经 c DDP处理后 ,用台盼蓝技术法检测 c DDP的 IC50 ,采用流式细胞仪、DAPI荧光染色、TUNEL 及 DNA电泳检测细胞凋亡。结果　c DDP在一定浓度范围内以浓度依赖的方式抑制骨肉瘤细胞生长 ,其 IC50 为 1.0 5 μg/ ml± 0 .2 5 μg/ ml。 0 .5～ 2 .0 μg/ mlc DDP处理骨肉瘤细胞后 ,流式细胞仪检测出凋亡峰 ,流式细胞光度计下可见明显的凋亡细胞形态特征 ,琼脂糖凝胶电泳出现 DNA梯形条带。结论　c DDP在体外可诱导骨肉瘤细胞凋亡 ,这种诱导细胞凋亡的能力是 c DDP疗效的基础 ,在对骨肉瘤的治疗中有着巨大的潜力。     

姜黄素联合顺铂对肺癌治疗的体外实验研究

目的探讨姜黄素（CUR）与化疗药（DDP）联合应用治疗肺癌的效应和可能机制。方法应用MTT试验检测CUR与DDP联用对人肺癌A549细胞增殖的影响，用流式细胞术检测CUR与DDP联用对人肺癌A549细胞周期和凋亡的影响。结果在一定的浓度范围内，随着姜黄素与顺铂均可抑制细胞生长，呈量-效关系。姜黄素与顺铂联用时，可以增强对A549细胞的增殖抑制作用。姜黄素和顺铂均可诱导A549细胞的凋亡，而且两者联用可增加A549细胞的凋亡率。姜黄素将细胞聚结在C2／M期并可诱导凋亡，顺铂将细胞聚结在s期并可诱导凋亡。结论姜黄素、顺铂均可抑制人肺腺癌A-549细胞的增殖、诱导细胞的凋亡，在一定浓度范围内，呈量-效关系，而且两者联合应用具有相加或协同作用。其效应可能是通过对细胞周期的影响来实现的。     

三氧化二砷与顺铂联用对肝癌HepG2细胞的作用

目的：探讨三氧化二砷（As2O3）与顺铂（CDDP）联合对肝癌细胞HepG2的作用。方法：应用普通光学显微镜、荧光显微镜、流式细胞仪分别观察As2O3、CDDP和两者联合应用时对HepG2细胞株的形态学改变和诱发凋亡率。结果：AO／EB荧光染色法显示在联合应用药物后,可看到较典型的细胞凋亡形态学改变。噻唑蓝（MTT）法检测各个浓度的As2O3与CDDP联合应用时,对HepG2细胞的生长抑制率均较单用相应一种药物时增强,而低浓度联合用药组合的生长抑制率增加尤为明显。结论：在体外Aa2O3和CDDP两种化疗药物联合应用具有明显的协同抗肝癌作用,显著抑制人肝癌细胞HepG2的细胞株的生长,并且具有明显的剂量时效关系。两药联用有显著的协同诱导肝癌细胞凋亡的作用。     

三种抗癌剂诱导肿瘤细胞凋亡与活性氧产生关系的研究

目的 探讨抗癌剂、活性氧、凋亡三者之间的关系,从而进一步探讨抗癌剂的作用及肿瘤细胞耐药机制以及活性氧在肿瘤发生上的意义。方法 用足叶乙甙（Vp16）、阿霉素（ADR）、顺铂（DDP0作用于K562细胞,用流式细胞仪检测三种抗癌剂在不同浓度、作用24h对K562细胞活性氧产生的影响及对凋亡的影响。同时用形态学方法观察凋亡细胞的形态改变。结果 ①Vp16、ADR、DDP三种抗癌剂均可通过激发K562细胞产生活性氧来诱导该细胞发生凋亡;②每种抗癌剂激发K562细胞产生活性氧及诱发该细胞凋亡各有其最适浓度(Vp16为5μg/ml,ADR为3μg/ml,DDP为μg/ml）及最佳作用时间（24h）。结论 流式细胞仪（FCM）测定细胞内活性氧产生状态及细胞凋亡情况,方法具有敏感、快速、简便、只需微量血、可除去坏死细胞碎片等优点。可用于抗癌剂的筛选,指导临床用药及选择敏感抗癌剂的有效剂量及最佳时间,并为探索治疗肿瘤新方法提供一个思路。     

The role of reactive oxygen species in tumor treatment

Reactive oxygen species (ROS) are by-products of aerobic metabolism and can also act as signaling molecules to participate in multiple regulation of biological and physiological processes. The occurrence, growth and metastasis of tumors, and even the apoptosis, necrosis and autophagy of tumor cells are all closely related to ROS. However, ROS levels in the body are usually maintained at a stable status. ROS produced by oxidative stress can cause damage to cell lipids, protein and DNA. In recent years, ROS have achieved satisfactory results on the treatment of tumors. Therefore, this review summarizes some research results of tumor treatments from the perspective of ROS in recent years, and analyzes how to achieve the mechanism of inhibition and treatment of tumors by ROS or how to affect the tumor microenvironment by influencing ROS. At the same time, the detection methods of ROS, problems encountered in the research process and solutions are also summarized. The purpose of this review is to provide a clearer understanding of the ROS role in tumor treatment, so that researchers might have more inspiration and thoughts for cancer prevention and treatment in the next stage.

This review provides a clear understanding of the ROS role in tumor treatment and some thoughts for potential cancer prevention.     

Radiation-induced red cell damage: role of reactive oxygen species

BACKGROUND: Cellular blood components are irradiated to prevent graft- versus-host disease in transfusion recipients at risk for this syndrome. Because gamma radiation can result in the production of reactive oxygen species, the role of reactive oxygen species was investigated in radiation-induced red cell damage. STUDY DESIGN AND METHODS: Whole blood from normal donors was exposed to various doses of t-butyl hydroperoxide (0-1 mM) and/or to gamma-radiation (0-50 Gy). Oxidative damage was assessed by the extent of lipid peroxidation (measured by thiobarbituric acid-reactive substances [TBARS]) and hemoglobin oxidation. Fresh blood was divided into three parts-one initially irradiated and stored, another stored with portions irradiated weekly, and a third stored without irradiation. TBARS and hemoglobin oxidation were measured weekly. RESULTS: As expected, t- butyl hydroperoxide induced TBARS formation and hemoglobin oxidation in a dose-dependent fashion. The gamma-radiation not only increased hemoglobin oxidation and TBARS formation, but also enhanced the t-butyl hydroperoxide effect on red cells. Red cell storage increased TBARS generation and hemoglobin oxidation in a time-dependent fashion. When radiation was administered either initially or after weekly storage, TBARS production and hemoglobin oxidation were increased over that measured in unirradiated paired controls. CONCLUSION: Gamma radiation at clinically used doses increases lipid peroxidation and hemoglobin oxidation in human red cells. The effect of gamma-radiation is accentuated by blood storage and induces damage independent of time of storage.     

铁过载诱导活性氧物质生成对骨髓造血功能影响的体外实验研究

目的 建立体外铁过载骨髓造血细胞模型,检验铁过载对细胞活性氧物质(ROS)水平的影响以及ROS升高对骨髓造血功能的影响。方法 在骨髓单个核细胞培养的过程中添加枸橼酸铁铵(FAC),使细胞铁过载,检验这一过程中细胞ROS水平、细胞凋亡水平、造血细胞集落形成和CD 34+细胞计数的变化。再用去铁胺(DFO)祛铁或抗氧化剂N-乙酰半胱氨酸(NAC)清除过多的ROS后,检测上述指标的变化。结果 ①在培养液中加入不同浓度FAC培养不同时间,发现骨髓造血细胞内可变铁池(LIP)水平升高,且具有时间和浓度依赖性,在含400μmoL/L FAC的培养液中培养24h时LIP水平达到最高。②在400μmol/L FAC浓度下,培养骨髓造血细胞24h后骨髓造血细胞内总的ROS、粒细胞和红细胞内ROS显著升高,分别为对照组的1.77、1.75和2.12倍。与对照组比较,DFO和NAC处理后均能明显降低细胞内ROS水平(P＜0.05)。③对骨髓细胞造血功能的检测发现FAC组细胞凋亡比例[(24.80±2.99)％]较对照组[(8.90±0.96)％]显著升高;造血细胞集落形成单位(CFUE、CFU-GM、BFU-E和CFU-mix)计数明显低于对照组(P值均＜0.05);CD34+细胞比例[(0.39±0.07)％]较对照组[(0.91±0.12)％]也显著降低。且这些损伤都可以通过DFO和NAC处理而部分恢复。结论 铁过载通过诱导ROS生成影响骨髓造血功能,这种损伤可以通过祛铁和抗氧化处理减轻。可能为治疗铁过载患者骨髓造血功能低下寻找新的靶点。     

The role of reactive oxygen species in thromboxane b2 generation by polymorphonuclear leukocytes

These studies evaluated further the relationship between the metabolism of reactive oxygen species (ROS) and prostaglandins in human granulocytes. Our experiments examined (1) the effects of several scavengers of ROS on thromboxane B2 (TXB2) production by zymosan-stimulated PMNs, (2) the capacity of the granulocytes of patients with chronic granulomatous disease (CGD) to produce TXB2, and finally (3) the generation of oxygen radicals in PMNs stimulated to produce TXB2 by the enzyme phospholipase A2. Our results confirm that both zymosan- and PMA-stimulated PMNs release increased amounts of TXB2. This enhanced production of TXB2 by normal PMNs could not be impaired and, in fact, appeared to be enhanced by scavengers of ROS. The PMNs of one patient with CGD produced TXB2 in an amount similar to those of healthy persons, whereas the TXB2 produced by the PMNs of a second patient was markedly increased. Finally, the enzyme phospholipase A2 stimulated TXB2 production in PMNs without stimulating the production of ROS. These data indicate that the activation of prostaglandin metabolism in PMNs is not dependent on the simultaneous production of ROS by these cells. However, the simultaneous production of ROS may be associated with an alteration of prostaglandin metabolism.     

Reactive oxygen species mediated apoptosis of esophageal cancer cells induced by marine triprenyl toluquinones and toluhydroquinones

Marine invertebrates, algae, and microorganisms are prolific producers of novel secondary metabolites. Some of these secondary metabolites have the potential to be developed as chemotherapeutic agents for the treatment of a wide variety of diseases, including cancer. We describe here the mechanism leading to apoptosis of esophageal cancer cell lines in the presence of triprenylated toluquinones and toluhydroquinones originally isolated from the Arminacean nudibranch Leminda millecra. Triprenylated toluquinone-induced and toluhydroquinone-induced cell death is mediated via apoptosis after a cell cycle block. Molecular events include production of reactive oxygen species (ROS), followed by induction and activation of c-Jun (AP1) via c-Jun-NH2-kinase-mediated and extracellular signal-regulated kinase-mediated pathways. Partial resistance to these compounds could be conferred by the ROS scavengers Trolox and butylated hydroxyanisol, a c-Jun-NH2-kinase inhibitor, and inhibition of c-Jun with a dominant negative mutant (TAM67). Interestingly, the levels of ROS produced varied between compounds, but was proportional to the ability of each compound to kill cells. Because cancer cells are often more susceptible to ROS, these compounds present a plausible lead for new antiesophageal cancer treatments and show the potential of the South African marine environment to provide new chemical entities with potential clinical significance.     

Up-regulation of reactive oxygen species (ROS) and resistance to Fas-mediated apoptosis in the C33A cervical cancer cell line transfected with IL-18 receptor.

Cervical cancer cells were transfected with a newly discovered interleukin (IL)-18 receptor to investigate the effect of endogenous IL-18 on the regulation of immune-related factors such as Fas (CD95/Apo-1)/Fas ligand and intercellular adhesion molecules. Transfection of the IL-18 receptor selectively induced a slight enhancement of the Fas via the up-regulation of intracellular reactive oxygen species and IL-18 in cervical carcinoma C33A cells, whereas there were no effects on the expression of p53, intercellular adhesion molecules-1 and Fas ligand. Neither IL-18 receptor transfection nor recombinant IL-18 enhanced interferon-gamma production in C33A cells. Thus, IL-18 receptor transfection induced IL-18 expression and enhanced intracellular reactive oxygen species and Fas expression in C33A cells in an interferon-gamma-independent pathway. However, treatment with agonistic anti-Fas antibody did not induce the apoptosis of C33A/IL-18 receptor transfectants, suggesting that either reactive oxygen species play a key role in resisting the Fas-induced apoptosis of C33A cells, or Fas was not functional. These results show that C33A/IL-18 receptor cells are resistant to the apoptosis and thus can survive against the immune surveillance and activated immune cells. Our results thus suggest that IL-18 and IL-18 receptor, together, may play a role in immunoregulation or in inflammation by augmenting the levels of IL-18 and reactive oxygen species in C33A cells.     

Characterization of chromatin modified with reactive oxygen species: recognition by autoantibodies in cancer

OBJECTIVES: To study the binding of chromatin modified with reactive oxygen species (ROS) with circulating autoantibodies present in cancer patients. DESIGN AND METHODS: Chromatin isolated from goat liver was modified by superoxide radical plus singlet oxygen and hydroxyl radicals. Sera from 47 patients with various types of cancers were tested for binding to native and modified chromatin by direct binding and competition ELISA. RESULTS: Maximum modification of thymine (54%) was found in case of chromatin modified with hydroxyl radical whereas by the combined action of singlet oxygen and superoxide anion radical, guanine was modified most (72%). Autoantibodies in cancer sera recognized modified chromatin in preference to native chromatin. Band shift assay with affinity-purified IgG from sera of cancer patients reiterated the results obtained with serum samples. CONCLUSION: Greater recognition of ROS-modified chromatin with the autoantibodies in cancer sera is indicative of reactive-oxygen-species-induced chromatin damage in cancer patients.     

Role of intracellular calcium ions and reactive oxygen species in apoptosis induced by ultrasound

Recently, we have reported that ultrasound (US)-induced apoptosis is due to inertial cavitation and that extracellular reactive oxygen species (ROS) generated by inertial cavitation are not directly correlated with the apoptosis (Honda et al. 2002). The molecular mechanism of apoptosis induced by US is not yet sufficiently clear. Here, we examine the role of intracellular calcium ions and the intracellular ROS on apoptosis induced by US. Human myelomonocytic lymphoma U937 cells were exposed to continuous 1-MHz US at an intensity of 4.9 W/cm(2) (I(SPTA)) in the presence of air, and changes of intracellular calcium ion concentration ([Ca(2+)]i) in individual cells by digital imaging, various flow cytometric analyses of endpoints of apoptosis (early apoptosis, secondary necrosis, loss of mitochondria membrane potential, superoxide formation, caspase-3 activation) and DNA fragmentation were explored. Furthermore, the effects of an intracellular calcium ion chelator (BAPTA-AM), an antioxidant (N-acetyl-L-cysteine, NAC), a calcium channel blocker (verapamil), Ca(2+)-free buffer and Levovist were also investigated. These results indicate that: 1. the mitochondria-caspase pathway and the Ca(2+)-dependent pathway play cardinal roles in apoptosis induced by US because BAPTA-AM partially inhibited DNA fragmentation, loss of mitochondria membrane potential and caspase-3 activation; 2. intracellular ROS generated from mitochondria, rather than extracellular ROS (which were directly produced by inertial cavitation in the medium), are involved in the regulation of apoptosis induced by US because addition of NAC after sonication showed effective suppression of the apoptosis; and 3. increase of [Ca(2+)]i appears to be due to nonspecific influx from outside the cells because verapamil is not effective and no increase of [Ca(2+)]i due to sonication could be observed in the Ca(2+)-free buffer.