manumycin诱导舌鳞癌Tca8113细胞凋亡的作用及机制

目的研究manumycin抑制Tca8113细胞的生长和诱导细胞凋亡的作用及机制。方法细胞毒作用以MTT法测定;凋亡细胞形态以Hoechst33258染色后荧光显微镜观察;凋亡率的检测以AnnexinV染色,流式细胞仪检测;细胞内活性氧(ROS)和线粒体跨膜电位(ΔΨm)分别用DCFH-DA和DiOC6荧光探针标记,流式细胞仪检测;蛋白质定量检测以Westernblot法。结果manumycin浓度依赖性抑制Tca8113细胞的生长,IC50为(11.33±0.63)μmol.L-1;manumycin可浓度和时间依赖性诱导Tca8113细胞的凋亡,过氧化物清除剂N-乙酰半胱氨酸能清除manumycin介导的细胞活性氧的增加,抑制线粒体跨膜电位降低及细胞凋亡。结论manumycin体外显著抑制舌鳞癌Tca8113的生长及诱导细胞凋亡,其机制可能通过激活线粒体依赖性凋亡通路有关。     

雷公藤红素通过活性氧诱导结肠癌SW620细胞凋亡

目的:探讨雷公藤红素(celestrol)诱导KRAS驱动的结肠癌SW620细胞产生凋亡的作用和机制。方法:四甲基偶氮唑蓝(MTT)法和台盼蓝拒染法检测细胞增殖;免疫印迹法检测蛋白表达;流式细胞仪和荧光显微镜检测细胞凋亡、细胞周期、线粒体膜电位;荧光显微镜检测细胞内活性氧水平(reactive oxygen species,ROS)。结果:雷公藤红素明显抑制SW620细胞的增殖活性;雷公藤红素下调SW620胞内的p-Akt、NF-κB、Survivin表达,激活caspase-7、caspase-3 和PARP;雷公藤红素增加SW620细胞内的ROS、降低线粒体膜电位、阻滞细胞周期于G₂/M期和诱导凋亡。抗氧化剂N-乙酰半胱氨酸(NAC)抑制雷公藤红素引起的上述作用。结论:通过诱导细胞内ROS的累积导致细胞内线粒体膜电位的下降进而触发细胞发生凋亡是雷公藤红素诱导SW620细胞凋亡的作用机制之一。     

D-氨基葡萄糖衍生物对Eca-109细胞内活性氧、线粒体膜电位的影响

目的：探讨2-（3-羧基-1-丙酰氨基）-2-脱氧-D-葡萄糖（2-[（3-carboxy-1-oxoprogy1）amino]-2-deoxy-D-Glucose,COPADG）诱导Eca-109细胞凋亡的机制。方法：不同浓度COPADG作用于人食管癌Eca-109细胞24h,检测Eca-109细胞的抑制率、凋亡率、细胞内活性氧（ROS）、线粒体跨膜电位。结果：Eca-109细胞凋亡率与COPADG浓度呈正相关（rs=1.0,P〈0.01）;Eca-109细胞线粒体膜电位水平与Eca-109细胞凋亡率相关（rs=1.0,P〈0.01）;ROS水平与Eca-109细胞凋亡率呈正相关（rs=1.0,P〈0.01）;ROS水平与Eca-109细胞线粒体膜电位水平呈负相关（rs=1.0,P〈0.01）。结论：COPADG可促进Eca-109细胞凋亡,提高Eca-109细胞内ROS水平,并降低线粒体膜电位水平。实验结果提示COPADG提高ROS水平,降低Eca-109细胞线粒体膜电位水平,启动细胞凋亡通路,促使Eca-109细胞凋亡,并且线粒体膜电位的下降是通过提高ROS实现的。     

北美海篷子三萜皂苷bigelovii E抑制mTOR信号通路诱导乳腺癌MCF-7细胞凋亡

目的探究北美海篷子的bigelovii E诱导乳腺癌MCF-7细胞凋亡的作用机制。方法使用MTT和集落克隆实验检测bigelovii E对肿瘤细胞增殖的抑制作用;Hoechst33258荧光染色观察bigelovii E作用后细胞形态的改变;流式细胞仪分析bigelovii E对细胞凋亡、细胞线粒体膜电位、细胞ROS水平的影响;Western blot分析线粒体凋亡通路及其调控蛋白表达量的变化。结果 Bigelovii E对乳腺癌细胞MCF-7最敏感;bigelovii E对正常细胞HLF-1毒性较低;bigelovii E抑制乳腺癌MCF-7的集落形成;Hoechst 33258荧光染色检测到bigelovii E诱导的细胞凋亡形态;流式细胞仪检测到bigelovii E引起细胞凋亡和细胞线粒体膜电位下降,并使细胞内ROS水平升高;Western blot表明bigelovii E可通过抑制m TOR磷酸化水平,下调其下游靶点p70S6K、4-EBP的磷酸化水平,进而上调Bax,下调Bcl-xl、Mcl-1蛋白表达,促进线粒体凋亡通路,降低线粒体膜电位,诱发ROS,导致线粒体功能损伤,最终诱导细胞凋亡。结论 Bigelovii E通过m TOR通路调控线粒体凋亡通路,诱导细胞凋亡,具有良好的抗肿瘤效果。     

D-氨基葡萄糖衍生物诱导Eca-109细胞凋亡的机制

目的探讨2-(3-羧基-1-丙酰氨基)-2-脱氧-D-葡萄糖{2-[(3-carboxy-1-oxoprogy1)amino]-2-deoxy-D-Glucose,CO-PADG}诱导Eca-109细胞凋亡的机制。方法不同浓度COPADG作用于人食管癌Eca-109细胞24h,检测Eca-109细胞的抑制率、凋亡率、细胞内活性氧(reactive oxygen spe-cies,ROS)、线粒体跨膜电位。结果Eca-109细胞凋亡率与COPADG浓度呈正相关,r=1.0,P<0.01;Eca-109细胞线粒体膜电位水平与Eca-109细胞凋亡率相关,r=1.0,P<0.01;ROS水平与Eca-109细胞凋亡率呈正相关,r=1.0,P<0.01;ROS水平与Eca-109细胞线粒体膜电位水平呈负相关,r=1.0,P<0.01。结论COPADG可促进Eca-109细胞凋亡,提高Eca-109细胞内ROS水平,并降低线粒体膜电位。实验结果提示COPADG提高ROS,降低Eca-109细胞线粒体膜电位启动细胞凋亡通路促使Eca-109细胞凋亡,并且线粒体膜电位的下降是通过提高ROS实现的。     

齿科合金活化线粒体途径诱导L929细胞凋亡的观察

目的：探讨齿科合金通过线粒体途径诱导L929细胞凋亡的作用机制。方法：选用临床常用齿科合金和中科院金属研究所研发的三种低镍合金的浸提液处理小鼠L929细胞,用MTT法检测细胞增殖：用荧光染料罗丹明123检测细胞线粒体跨膜电位;Western-Blot检测线粒体凋亡通路相关蛋白的表达。结果：齿科合金作用L929细胞48h后,细胞线粒体跨膜电位降低,伴有细胞色素C从线粒体到胞浆的释放。结论：齿科合金通过活化线粒体信号转导途径引起线粒体跨膜电位下降与细胞色素C的释放可能是合金诱导L929细胞凋亡途径之一。     

格列苯脲通过激活ROS-JNK通路发挥抗肿瘤作用

目的：探讨ATP敏感性钾通道（KATP）阻断剂格列本脲对胃癌细胞株MGC-803的抗肿瘤作用及相关机制。方法：应用MTT、Hoechst法分别检测格列本脲对细胞活力和凋亡的影响：应用Western．blot、RT-PCR等方法检测MGC-803细胞KATP亚基的表达、线粒体细胞色素C和凋亡诱导因子（AIF）的释放、JNK,Akt的磷酸化,以及NADPH氧化酶催化亚基gp91的表达;应用H2DCFDAROS荧光探针检测ROS和O2-的产生,氧电极法测定线粒体呼吸功能。结果：MGC-803细胞表达由kir6．2和SUR1亚基构成的KATP通道。格列苯脲可降低MGC-803细胞活力、促进其凋亡;格列苯脲能促进NADPH氧化酶源性和线粒体源性ROS的生成,进而活化JNK激酶,并降低线粒体膜电位、促进细胞色素C和凋亡诱导因子（AIF）的释放,触发线粒体通路介导的细胞凋亡;抗氧化剂N-乙酰半胱氨酸（NAC）可抑制格列苯脲降低MGC-803细胞活力、诱发细胞凋亡,以及活化JNK的作用;进一步的研究显示,格列苯脲可显著降低野生型MEF细胞的活力、促进其凋亡,并能抑制抗凋亡的Akt信号通路,但不影响JNK1和JNK2-MEF细胞的存活。结论：KATP,通道阻断剂格列苯脲可通过促进ROS的生成,激活JNK、抑制Akt信号通路,触发线粒体通路介导的细胞凋亡,从而发挥其抗肿瘤作用。研究结果提示,KATP通道可能是发展抗肿瘤药物的有效靶标。     

碲化镉量子点对血管平滑肌细胞的损伤作用

目的观察碲化镉量子点(CdTe QD)在体外对大鼠血管平滑肌细胞(VSMC)的损伤作用。方法CdTe QD(0.01~100 mg·L~(-1))与原代培养的大鼠胸/腹主动脉平滑肌细胞共孵育24 h,MTT法检测CdTe QD对细胞存活的抑制;钙黄绿素乙酰甲酯(calcein-AM)荧光染色观察CdTe QD对VSMC细胞活性的影响;DCFH-DA和JC-1染色、流式细胞术检测CdTe QD作用后细胞内活性氧(ROS)含量和线粒体膜电位的变化;流式细胞术检测细胞凋亡;Western蛋白印迹法检测BCL-2和BAX蛋白表达变化。结果 MTT结果显示,CdTe QD 0.01~100 mg·L~(-1)作用VSMC细胞24 h,细胞存活率降低(P<0.01),24 h IC_(50)值为25.60 mg·L~(-1)。Calcein-AM荧光检测发现,CdTe QD 0.1~25 mg·L~(-1)作用后,VSMC细胞活性下降。DCFH-DA染色结果显示,CdTe QD 0.1~25 mg·L~(-1)使细胞内ROS逐渐增加(P<0.05,P<0.01);JC-1染色结果表明,VSMC线粒体膜电位呈浓度依赖性降低(r=0.903,P<0.01)。Western蛋白印迹结果表明,CdTe QD诱导抗凋亡蛋白BCL-2表达显著降低(P<0.01),促凋亡蛋白BAX表达显著上升(P<0.01);流式细胞术FITC-AnnexinⅤ染色结果显示,CdTe QD 0.1~25 mg·L~(-1)作用24 h能显著促进VSMC细胞凋亡(P<0.05,P<0.01)。结论CdTe QD可诱导VSMC细胞凋亡,其机制可能与胞内ROS含量上升和线粒体介导的凋亡通路激活相关。     

右美托咪定对高氧诱导的人肺泡上皮细胞线粒体损伤及HIF-1α/ERK通路的影响

摘要：目的:观察右美托咪定（Dex）对高氧诱导的人肺泡上皮细胞HPAEpiC线粒体损伤及缺氧诱导因子-1α（HIF-1α）/细胞外信号调节激酶（ERK）通路的影响。方法:培养HPAEpiC细胞,分为对照（Control）组、高氧模型（Hyperoxia）组、高氧和药物处理（Hyperoxia+Dex）组、高氧和HIF-1α激动剂处理（Hyperoxia+DMOG）组以及高氧和药物、HIF-1α抑制剂处理（Hyperoxia+Dex+YC-1）组。采用活性氧（ROS）检测试剂盒和MitoSOXTM?Red荧光染色分别检测细胞中和线粒体中ROS含量;线粒体膜电位检测试剂盒（JC-1法）检测线粒体膜电位;流式细胞仪检测细胞凋亡率;Western blot检测细胞中HIF-1α/ERK通路相关蛋白及核呼吸因子-1（NRF-1）蛋白水平。结果:与Control组比较,Hyperoxia组HPAEpiC细胞中和线粒体中ROS含量升高,线粒体膜电位降低,细胞凋亡率增高,细胞中HIF-1α、p-ERK和NRF-1蛋白水平降低（P<0.05）。与Hyperoxia组比较,Hyperoxia+Dex组和Hyperoxia+DMOG组HPAEpiC细胞中和线粒体中ROS含量降低,线粒体膜电位升高,细胞凋亡率降低,细胞中HIF-1α、p-ERK和NRF-1蛋白水平升高（P<0.05）。与Hyperoxia+Dex组比较,Hyperoxia+DMOG组和Hyperoxia+Dex+YC-1组HPAEpiC细胞中和线粒体中ROS含量升高,线粒体膜电位降低,细胞凋亡率增高,细胞中HIF-1α、p-ERK和NRF-1蛋白水平降低（P<0.05）。结论:Dex可能通过激活HIF-1α/ERK通路减轻高氧诱导的人肺泡上皮细胞线粒体损伤。     

瓜子金皂苷丙对MPP~+诱导PC12细胞凋亡的抑制作用

目的观察瓜子金皂苷丙对MPP+诱导的PC12细胞凋亡的影响,并且探讨其作用机制。方法采用MTT法检测细胞存活率,碘化丙啶染色流式细胞术(FCM)检测PC12细胞凋亡,Western blotting检测Bax和Bcl-2蛋白的表达,罗丹明123染色FCM检测细胞线粒体膜电位(ΔΨm),荧光酶标仪检测细胞内活性氧(R0S)的含量。结果不同浓度MPP+作用PC12细胞24 h后,细胞存活率显著下降(P<0.01),细胞凋亡明显,凋亡相关蛋白Bcl-2/Bax比之下降,线粒体膜电位降低,细胞内ROS显著增加。与MPP+处理组相比,瓜子金皂苷丙10μmol·L-1组,细胞存活率显著升高(P<0.01);细胞凋亡率下降(P<0.01);Bcl-2/Bax比率增加(P<0.01),线粒体膜电位上升(P<0.01),ROS含量减少。结论瓜子金皂苷丙可以抑制MPP+诱导的PC12细胞凋亡,其作用机理可能与上调Bcl-2和下调Bax蛋白的表达,维持线粒体正常膜电位,稳定线粒体功能,清除R0S有关。     

CR108, a novel vitamin K3 derivative induces apoptosis and breast tumor inhibition by reactive oxygen species and mitochondrial dysfunction

Vitamin K3 derivatives have been shown to exert anticancer activities. Here we show a novel vitamin K3 derivative (S)-2-(2-hydroxy-3-methylbutylthio)naphthalene-1,4-dione, which is named as CR108 that induces apoptosis and tumor inhibition through reactive oxygen species (ROS) and mitochondrial dysfunction in human breast cancer. CR108 is more effective on the breast cancer cell death than other vitamin K3 derivatives. Moreover, CR108 induced apoptosis in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. CR108 caused the loss of mitochondrial membrane potential, cytochrome c released from mitochondria to cytosol, and cleaved PARP proteins for apoptosis induction. CR108 markedly increased ROS levels in breast cancer cells. N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the CR108-induced ROS levels, mitochondrial dysfunction and apoptosis. Interestingly, CR108 increased the phosphorylation of p38 MAP kinase but conversely inhibited the survivin protein expression. NAC treatment prevented the activation of p38 MAP kinase and rescued the survivin protein levels. SB202190, a specific p38 MAP kinase inhibitor, recovered the survivin protein levels and attenuated the cytotoxicity of CR108-treated cells. Furthermore, CR108 inhibited the xenografted human breast tumor growth in nude mice. Together, we demonstrate that CR108 is a novel vitamin K3 derivative that induces apoptosis and tumor inhibition by ROS production and mitochondrial dysfunction and associates with the phosphorylation of p38 MAP kinase and the inhibition of survivin in the human breast cancer.     

Chelerythrine hydrochloride inhibits proliferation and induces mitochondrial apoptosis in cervical cancer cells via PI3K/BAD signaling pathway

Cervical cancer is the fourth most common female cancer worldwide, and drug targeted therapy plays a crucial role in delaying the progression of cervical carcinoma. Chelerythrine hydrochloride (CHE) is a natural alkaloid, which is a focal point in anti-tumor research. In this study, we investigated the effect of CHE on HeLa cells by using MTT assay, RTCA, and colony formation assay. In addition, the flow cytometric analysis, immunofluorescence staining assay and western blot analysis were performed to study the mechanism of CHE. The results showed that CHE exhibited a significant inhibitory effect in HeLa cells, and it could suppress the expression of PI3K subunits in a dose-dependent manner. Moreover, we found that the treatment of CHE further restrained the downstream AKT/mTOR and PKCα signaling. In addition, CHE induced mitochondrial apoptosis of HeLa cells by regulating the BCL-2 family member's expression. Immunofluorescence staining assay indicated that AIF and Cytochrome C were translocated from mitochondria to cytoplasm or nucleus, and notable changes in mitochondrial morphology of HeLa cells were also observed. Finally, the aberrant expression of CHE led to the mitochondrial apoptosis by upregulating the expression of APAF1, Cleaved-Caspase9, Cleaved-Caspase3, and Cleaved-PARP. In summary, CHE suppresses the proliferation of HeLa cells by trigging the mitochondrial apoptosis through the PI3K/BAD signaling pathway.     

Strategic formulation of apigenin-loaded PLGA nanoparticles for intracellular trafficking,DNA targeting and improved therapeutic effects in skin melanoma in vitro

The aim of the present study was the evaluation of anti-proliferative potentials of apigenin (Ap), (a dietary flavonoid) loaded in poly (lactic-co-glycolide) nanoparticles (NAp) in A375 cells in vitro. NAp was characterized for particle size, morphology, zeta potential, drug release and encapsulation. Cellular entry and intracellular localization of NAp were assessed by transmission electron and confocal microscopies. Circular dichroic spectral analysis and stability curve for Gibb's free energy of dsDNA of A375 cells were also analyzed. DNA fragmentation, intracellular ROS accumulation, superoxide-dismutase activity, intracellular glutathione-reductase content and mitochondrial functioning through relevant markers like mitochondrial transmembrane potential, ATPase activity, ATP/ADP ratio, volume changes/swelling, cytochrome-c release, expressions of Apaf-1, bax, bcl-2, caspase-9, 3, and PARP cleavage were analyzed. NAp produced better effects due to their smaller size, faster mobility and site-specific action. Photostability studies revealed that PLGA encapsulations were efficient at preserving apigenin ultraviolet-light mediated photodegradation. NAp readily entered cancer cells, could intercalate with dsDNA, inducing conformational change. Corresponding increase in ROS accumulation and depletion of the antioxidant enzyme activities exacerbated DNA damage, mediating apoptosis through mitochondrial dysfunction. Overall results indicate that therapeutic efficacy of NAp may be enhanced by PLGA nanoparticle formulations to have better ameliorative potentials in combating skin melanoma.     

Ellipticine induces apoptosis in human endometrial cancer cells: the potential involvement of reactive oxygen species and mitogen-activated protein kinases

Ellipticine, an alkaloid isolated from Apocyanaceae plants, has been shown to exhibit antitumor activity in several human malignant tissues including breast, thyroid, and ovarian cancers. The antitumor activity of ellipticine is thought to be primarily mediated by the induction of DNA damage through the inhibition of topoisomerase II and formation of DNA adducts. The human endometrium is known to express topoisomerase II. However, the apoptogenic activity of ellipticine and the mechanisms underlying its action have not been investigated in endometrial cancer cells. In the present study, exposure to ellipticine (1-10μM) was shown to induce apoptosis in RL95-2 human endometrial cancer cells. Ellipticine-induced cell death was associated with the accumulation of cells in the G2/M phase of the cell cycle and was accompanied by depolarization of the mitochondrial membrane potential, release of cytochrome c and apoptosis-inducing factor (AIF) from the mitochondrial membrane, and caspase activation. The production of intracellular reactive oxygen species (ROS) was increased and sustained at high levels during ellipticine treatment. Subsequent to ROS accumulation, extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) were activated in ellipticine-treated cells. Release of AIF from the mitochondria appeared to be affected by caspases, ROS, and ERK. The present data show that the treatment of RL95-2 cells with ellipticine induces apoptosis, ellipticine-induced apoptosis is mediated by ROS and the activation of MAPKs, and release of AIF is involved in a caspase-independent pathway. These results demonstrate the potential of ellipticine as a therapeutic strategy for the treatment of human endometrial cancers.     

Emodin induces human T cell apoptosis in vitro by ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction

Aim:

To elucidate the molecular mechanisms underlying the immunosuppressive effects of emodin isolated from Rheum palmatum L.

Methods:

Human T cells were isolated from the peripheral venous blood of 10 healthy adult donors. Cell viability was analyzed with MTT assay. AO/EB and Annexin V/PI staining and DNA damage assay were used to detect cell apoptosis. Fluorescence staining was used to detect the levels of ROS, the mitochondrial membrane potential and intracellular Ca²⁺. Colorimetry was used to detect the levels of MDA and total SOD and GSH/GSSG ratio. The expression and activity of caspase-3, -4, and -9 were detected with Western blotting and a fluorometric assay. Western blotting was also used to detect the expression of Bcl-2, Bax, cytochrome C, and endoplasmic reticulum (ER) markers.

Results:

Emodin (1, 10, and 100 μmol/L) inhibited the growth of human T cells and induced apoptosis in dose- and time dependent manners. Emodin triggered ER stress and significantly elevated intracellular free Ca²⁺ in human T cells. It also disrupted mitochondrial membrane potential, and increased cytosolic level of cytochrome C, and the levels of activated cleavage fragments of caspase-3, -4, and -9 in human T cells. Furthermore, emodin significantly increased the levels of ROS and MDA, inhibited both SOD level and GSH/GSSG ratio in human T cells, whereas co-incubation with the ROS scavenger N-acetylcysteine (NAC, 20 μmol/L) almost completely blocked emodin-induced ER stress and mitochondrial dysfunction in human T cells, and decreased the caspase cascade-mediated apoptosis.

Conclusion:

Emodin exerts immunosuppressive actions at least partly by inducing apoptosis of human T cells, which is triggered by ROS-mediated ER stress and mitochondrial dysfunction.     

A novel synthetic analog of militarin,MA-1 induces mitochondrial dependent apoptosis by ROS generation in human lung cancer cells

A synthetic Militarin analog-1[(2R,3R,4R,5R)-1,6-bis(4-(2,4,4-trimethylpentan-2-yl)phenoxy) hexane-2,3,4,5-tetraol] is a novel derivative of constituents from Cordyceps militaris, which has been used to treat a variety of chronic diseases including inflammation, diabetes, hyperglycemia and cancers. Here, we report for the first time the synthesis of Militarin analog-1 (MA-1) and the apoptotic mechanism of MA-1 against human lung cancer cell lines. Treatment with MA-1 significantly inhibited the viability of 3 human lung cancer cell lines. The inhibition of viability and growth in MA-1-treated A549 cells with an IC₅₀ of 5 μM were mediated through apoptosis induction, as demonstrated by an increase in DNA fragmentation, sub-G₀/G₁-DNA fraction, nuclear condensation, and phosphatidylserine exposure. The apoptotic cell death caused mitochondrial membrane permeabilization through regulation of expression of the Bcl-2 family proteins, leading to cytochrome c release in a time-dependent manner. Subsequently, the final stage of apoptosis, activation of caspase-9/-3 and cleavage of poly (ADP ribose) polymerase, was induced. Furthermore, A549 lung cancer cells were more responsive to MA-1 than a bronchial epithelial cell line (BEAS-2B), involving the rapid generation of reactive oxygen species (ROS), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) activation. The pharmacological inhibition of ROS generation and JNK/p38 MAPK exhibited attenuated DNA fragmentation in MA-1-induced apoptosis. Oral administration of MA-1 also retarded growth of A549 orthotopic xenografts. In conclusion, the present study indicates that the new synthetic derivative MA-1 triggers mitochondrial apoptosis through ROS generation and regulation of MAPKs and may be a potent therapeutic agent against human lung cancer.     

Ascorbic acid induces Ca~(2+)-dependent necrosis mediated by reactive oxygen species in colorectal cancer cells

OBJECTIVE Ascorbic acid(AA), commonly known as vitamin C, is a small molecular widely distributed in in food and traditional herbs. Recently, there are some literatures reported that high concentration AA could selectively kill the cancer cells but not the normal cells. This study was designed to explore the underlying mechanisms. METHODS Colorectal cancer line cells were cultured and treated with AA. The cytotoxic, intracellular ATP level, reactive oxygen species, calcium, were determined with commercial kits and fluorescent probes. RESULTS High concentration of AA induced cell death in HCT116 and HT29 colorectal cancer cells in concentration-and time-dependent manner. AA treatment induced ATP decrease, LDH release, cell swollen and loss of plasma membrane integrity. Pharmacological inhibitors for apoptosis, necroptosis, autophagy, pyroptosis and oncosis showed no effect on AA-induced cell death. Furthermore, ROS level increase and intracellular calcium(Ca~(2+) ) accumulation were observed after AA treatment. ROS scavenger N-acetyl cysteine(NAC), intracellular calcium chelator BAPTA-AM and intracellular calcium inhibitor 2-aminoethoxydiphenyl borate(2-APB) could attenuate the cell death induced by AA. NAC could attenuate both ROS increase and intracellular Ca~(2+) accumulation induced by AA, while BAPTA-AM could only attenuate intracellular Ca~(2+) accumulation. In addition, high concentration AA induced mitochondrial damage and mitochondrial ROS generation. CONCLUSION AA induces Ca~(2+) -dependent programed necrosis mediated by ROS. Our study provided new insights into high concentration AA induced cell death in human colon cancer cells.     

A novel 7‐azaisoindigo derivative‐induced cancer cell apoptosis and mitochondrial dysfunction mediated by oxidative stress

This research focused on a novel 7‐azaisoindigo derivative [namely N¹‐(n‐butyl)‐7‐azaisoindigo, 7‐AI‐b], and investigated its molecular antitumor mechanism by exploring the means of cell death and the effects on mitochondrial function. 7‐AI‐b inhibited cancer cell proliferation in a dose‐ and time‐dependent way. The morphological and nuclei changes in H₂B‐GFP‐labeled HeLa cells were observed using a live cell system. The results suggested that cell death induced by 7‐AI‐b is closely related to apoptosis. 7‐AI‐b induced release of cytochrome C from mitochondria to cytosol and activation of caspase‐3, showing that the apoptosis is mediated by the mitochondrial pathway. Furthermore, our data indicated that 7‐AI‐b triggers apoptosis through reactive oxygen species (ROS): cellular ROS levels were increased after 3 h exposure of 7‐AI‐b, which was reversed by the ROS scavenger N‐acetyl‐l ‐cysteine. As a consequence, 7‐AI‐b‐mediated cell death, mitochondrial transmembrane potential collapse and ATP level were partly blocked by N‐acetyl‐l ‐cysteine. Further study showed that 7‐AI‐b could induce mitochondrial dysfunction: collapse of the mitochondrial transmembrane potential and reduction of intracellular ATP level. In summary, the novel synthesized 7‐AI‐b was demonstrated to be effective in killing cancer cells via an ROS‐promoted and mitochondria‐ and caspase‐dependent apoptotic pathway. Copyright © 2010 John Wiley & Sons, Ltd.     

Induction of apoptosis by chelerythrine chloride through mitochondrial pathway and Bcl-2 family proteins in human hepatoma SMMC-7721 Cell

The objective of this study was to evaluate the antitumor activity of chelerythrine chloride (CHE) and investigate its potential apoptotic induction mechanism in SMMC-7721 cells. Our results suggested that the proliferation of SMMC-7721 cells was inhibited by CHE in a time and dose dependent manner, with a significant accumulation in S phase, and the cells exhibited typical apoptotic features. Moreover, CHE remarkably induced apoptosis by disruption of the mitochondrial membrane potential, release of Cyt-c, activation of caspase-3, and cleavage of poly-ADP-ribose polymerase in a dose dependent manner. Furthermore, the expression of Bcl-xl was downregulated while Bax and Bid expression was upregulated, and no variation was found for Bcl-2. These results indicated that CHE may play an important role in suppression of tumor growth by inducing apoptosis in human hepatoma cells via the activation of a mitochondrial pathway and regulating the expression of Bcl-2 family proteins.