C2-神经酰胺对线粒体膜间隙蛋白释放影响研究

目的 探讨外源性C2-神经酰胺对线粒体膜间隙蛋白释放的影响.方法 12.5、25和50μmol/L C2-神经酰胺作用人结肠癌HT-29细胞24 h,采用Mitochondrial/Cytosol Fractionation试剂盒分离细胞线粒体和细胞液,应用Western Blotting方法检测线粒体和细胞液中Cyt c、Smac和HtrA2蛋白表达水平.结果 C2-神经酰胺作用细胞后,Cyt c、Smae和HtrA2总蛋白的表达水平未见明显改变,但随着C2-神经酰胺浓度增加,Cyt c、Smac和HtrA2从线粒体释放入细胞液中的水平逐渐增加,具有剂量-效应关系.Caspases抑制剂存在下,50 μrnol/L C2-神经酰胺仍能使Cyt c和HtrA2从线粒体释放入胞液中,但Smac却不能释放.结论 C2-神经酰胺能通过Caspases依赖和非依赖的方式促进线粒体膜间隙蛋白的释放入细胞液,诱导细胞凋亡作用.     

神经酰胺诱导结肠癌HT-29细胞凋亡与线粒体膜电位变化的关系

目的 探索神经酰胺(C2-ceramide,C2-cer)对人结肠癌HT-29细胞增殖、线粒体膜电位改变、细胞凋亡等的影响.方法 正常培养的HT-29细胞分为C2-cer处理组与非处理组,用MTT法、流式细胞仪等方法观察不同时间C2-cer对细胞增殖、线粒体膜电位改变、细胞凋亡等的影响.结果 加入C2-cer可引起HT-29细胞线粒体膜电位 (△Ψm)下降,细胞增殖受抑,并导致细胞凋亡.上述效应与C2-cer剂量以及作用时间相关.结论 C2-cer可导致HT-29细胞线粒体膜电位(△Ψm)下降,抑制细胞增殖,诱导细胞凋亡.     

槲寄生凝集素对人结肠癌HT-29细胞增殖及凋亡的影响

目的：研究槲寄生凝集素对人结肠癌HT-29细胞增殖和凋亡的影响。方法：采用MTT法观察槲寄生凝集素对体外培养的人结肠癌HT-29细胞增殖的影响,运用基因组DNA电泳观察凋亡特征性“梯状”条带及原位末端标记法检测槲寄生凝集素诱导HT-29细胞凋亡。结果：槲寄生凝集素对体外培养的人结肠癌HT-29细胞抑制作用表现出时间和剂量依赖性。基因组DNA琼脂糖凝胶电泳显示槲寄生凝集素作用于HT-29细胞后出现凋亡细胞特有的DNA阶梯状条带。原位末端标记法检测显示槲寄生凝集素可明显诱导HT-29细胞的凋亡。结论：槲寄生凝集素可抑制人结肠癌HT-29细胞生长并诱导其凋亡。     

C2-神经酰胺诱导大鼠脑胶质瘤细胞C6的早期凋亡作用

目的 :研究外源性C2 神经酰胺对大鼠脑胶质瘤细胞C6活力的抑制作用以及早期凋亡诱导作用。方法 :采用MTT法测定C2 神经酰胺对C6细胞活力的影响 ;倒置荧光显微系统观察细胞形态变化 ;An nexinⅤ PI双染色流式细胞术对细胞早期凋亡进行定量分析。结果 :C2 神经酰胺可显著抑制C6细胞的活力 ,IC50 为 2 .2× 10 -5mol·L-1;荧光染色可见核浓染等细胞凋亡特征形态改变 ;流式细胞术分析表明C2 神经酰胺可诱导C6细胞发生早期凋亡作用 ,且凋亡百分率呈时间及浓度依赖性 ,C2 神经酰胺 2× 10 -5mol·L-1处理 2 4h后平均早期凋亡率高达4 9 .3%。结论 :C2 神经酰胺主要通过诱导早期细胞凋亡对大鼠脑胶质瘤C6细胞发挥细胞毒作用 ,提高神经酰胺水平有望成为肿瘤化疗的新途径。     

活性氧介导5, 7- 二甲氧基黄酮诱导结肠癌HT- 29 细胞凋亡

目的研究5,7-二甲氧基黄酮（5,7-dimethoxyflavone,DMF）诱导体外培养人结肠癌HT-29细胞凋亡作用及其机制。方法以HT-29细胞为研究对象,利用ELISA法测定细胞DNA断裂,流式细胞术检测细胞凋亡率和活性氧（ROS）水平。结果DMF激活HT-29细胞ROS生成（P〈0．05）,呈浓度依赖性。DMF以浓度依赖方式促进HT-29细胞DNA断裂（P〈0．05）,同时,诱导细胞凋亡率增高（P〈0．05）;10μmol．L^-1N-乙酰半胱氨酸预孵育30min,能有效阻断DMF诱导ROS生成和细胞凋亡作用（P〈0．05）。结论DFM通过促进结肠癌HT-29细胞ROS生成诱导细胞凋亡。     

槲皮素诱导人结肠癌细胞HT-29凋亡的机制研究

目的 探讨槲皮素在体外对人结肠癌HT-29细胞凋亡的影响,并对其诱导机制进行研究。方法 体外常规培养HT-29细胞,随机设定对照组和高、中、低剂量（32,16,8 μg·mL^-1）槲皮素组,药物干预24 h后,采用CCK-8试剂盒检测槲皮素对HT-29细胞增殖的影响,倒置显微镜观察细胞形态变化,蛋白免疫印迹法检测MKK3/6/p38信号通路蛋白表达,并同时检测凋亡相关蛋白Caspase 8、Caspase 3、Bax及Bcl-2的表达水平。结果 槲皮素干扰HT-29细胞24 h后,CCK-8实验发现槲皮素可明显抑制细胞的增殖;形态学观察发现细胞密度明显减小、细胞体积变小;免疫印迹实验发现槲皮素可诱导MKK3/6及p38的磷酸化,进而促进促凋亡蛋白Caspase8、Caspase3及Bax的表达,抑制了抗凋亡蛋白Bcl-2的表达。结论 槲皮素可诱导人结肠癌HT-29细胞凋亡,其作用机制可能与激活MKK3/6/p38信号通路有关。     

小白菊内酯对结肠癌细胞凋亡的影响及其机制研究

目的观察小白菊内酯（PN）对结肠癌HT-29细胞凋亡的影响并分析其可能机制。方法不同浓度PN作用于结肠癌HT-29细胞,以流式细胞仪（FCM）检测细胞凋亡;Western Blot法检测核转录因子（NF）-B亚单位pso、环氧化酶（cox-2）蛋白表达的改变;ELISA法测定前列腺素E2（PGE2）浓度。结果PN能诱导结肠癌细胞的凋亡,且随PN浓度的增加,结肠癌HT-29细胞的凋亡率增加;而NF-κB亚单位p50、COX-2蛋白表达减少,同时PGE2浓度降低。结论PN可诱导结肠癌HT-29细胞凋亡,其机制可能与其能下调NF-κB、COX-2蛋白表达并减少PGE2生成有关。     

水通道蛋白-8对结肠癌细胞凋亡及凋亡抑制蛋白的影响

目的：通过表达水通道蛋白-8（AQP-8）真核表达载体,观察水通道蛋白AQP-8对HT-29细胞凋亡及凋亡抑制蛋白（ IAPs）表达的影响。方法取对数生长期的人结肠癌HT-29细胞株用于实验。构建AQP-8真核表达载体并转染HT-29细胞,通过Western blot检测GFP-AQP-8转染效率;采用MTT法检测各组细胞增殖抑制率;流式细胞术检测细胞凋亡率;通过Real Time-PCR和Western blot检测转染GFP-AQP-8的HT-29细胞凋亡抑制蛋白（ IAPs）家族成员c-IAP1、c-IAP2、XIAP、NIAP、Survivin和Livin表达水平。结果 Western blot结果显示,GFP-AQP-8转染后结肠癌HT-29细胞AQP-8基因表达显著上调（ P ＜0{．05）。 MTT分析结果显示,转染了GFP-AQP-8的结肠癌HT-29细胞增殖抑制率显著增加（ P ＜0．05）;流式细胞分析发现,转染了GFP-AQP-8的HT-29细胞凋亡率显著增加（ P ＜0．05）。Real Time-PCR和Western blot结果显示,与转染GFP-N1的阴性对照组相比较,转染了GFP-AQP-8的HT-29细胞c-IAP1、c-IAP1、XIAP、Livin和Survivin的mRNA和蛋白表达水平下降（ P ＜0．05）,NIAP表达变化不明显（ P ＜0．05）。结论过表达AQP-8可抑制HT-29细胞生长,并能通过下调c-IAP1、c-IAP1、XIAP、Livin和Survivin表达诱导HT-29细胞凋亡。     

CpGODN结合CD28/CD80单抗活化T细胞对结肠癌细胞的杀伤作用

目的研究联合应用CpGODN和CD28/CD80单抗共刺激健康人外周血T淋巴细胞(PBLs)在体外对结肠癌细胞株HT-29的杀伤作用,为结直肠癌的过继免疫治疗可能性提供参考。方法PBLs的分离与体外培养;CD28/CD80共刺激活化PBLs;用含共刺激活化PBMC或/和CpGODN的100ml/L胎牛血清的RPMI1640培养基培养细胞,测生长曲线。MTT法检测活化细胞的体外淋巴细胞毒作用,并用电镜观察效应细胞杀伤的结肠癌细胞超微结构及用流式细胞仪检测结肠癌细胞的相关凋亡情况。结果CpGODN本身对HT-29细胞有一定的杀伤作用(P<0.05),CD28/CD80共刺激活化PBLs在体外对结肠癌细胞株HT-29杀伤作用明显(P<0.01),CpGODN与CD28/CD80共刺激活化PBLs合用,对HT-29的杀伤作用显著大于CpGODN单独对HT-29细胞杀伤作用(抑制率92.31%vs68.00%,P<0.01),亦大于单独应用CD28/CD80共刺激活化PBMC对HT-29细胞的杀伤作用(P<0.05),CpGODN增加了HT-29细胞对共刺激活化PBMC的敏感性。电镜结果显示,效应细胞作用24h肿瘤细胞就部分发生坏死,部分肿瘤细胞可见凋亡。流式细胞仪检测效应细胞HT-29细胞72h明显凋亡。结论联合应用CpGODN可以提高单抗协同诱导的效应细胞对结肠癌细胞株HT-29杀伤作用,而坏死细胞进一步增多说明效应细胞是通过诱导肿瘤细胞坏死及细胞凋亡两条途径来实现抑制杀伤作用从而说明活化的淋巴细胞杀伤结肠癌细胞是通过坏死及凋亡实现的。     

塞来昔布对结肠癌HT-29细胞生长及骨桥蛋白表达的影响

目的 观察塞来昔布对结肠癌HT-29细胞的生长、凋亡和骨桥蛋白(OPN)表达的影响.方法 体外培养结肠癌HT-29细胞分为对照组和不同浓度塞来昔布干预组.MTT法检测细胞增殖抑制情况,流式细胞术分析细胞的凋亡,RT-PCR和免疫组化分析细胞中的OPN mRNA与OPN蛋白表达变化.结果 塞来昔布对HT-29细胞增殖有明显的时间与浓度依赖性抑制作用(P＜0.01).塞来昔布能诱导HT-29细胞凋亡,塞来昔布15、30和50μmol/L的细胞凋亡率分别为28.2％、32.8％和33.1％,均明显高于对照组的26.0％ (P＜0.05).药物干预组OPN mRNA及OPN蛋白表达明显低于对照组(P＜0.01).结论 塞来昔布可能通过抑制OPN表达而抑制结肠癌HT-29细胞的增殖,诱导其凋亡.     

Prevalence of necrosis in C2-ceramide-induced cytotoxicity in NB16 neuroblastoma cells

The mechanism of cell death triggered by C2-ceramide was investigated using the NB16 neuroblastoma cell line. Treatment of NB16 cells with 20 microM C2-ceramide for 20 h resulted in approximately 75% loss of cell viability, but only 25% of cells were scored as apoptotic based on terminal deoxynucleotidyl transferase nick-end labeling. Ultrastructural analysis revealed early development of necrotic cytoplasmic vacuolization. After 20 h of treatment with C2-ceramide, the majority of cells possessed necrotic morphology with pronounced cytoplasmic vacuolization and without any nuclear changes, although a quarter of the cell population also exhibited clear perinuclear chromatin condensation characteristic of apoptosis. Flow cytometric analysis of cells labeled with both annexin V and propidium iodide showed the rapid accumulation of C2-ceramide-treated cells in the necrotic/late apoptotic fraction. In contrast, cells treated with tumor necrosis factor alpha plus cycloheximide (TNFalpha + CHX) first appeared in the early apoptotic fraction and then accumulated in the necrotic/late apoptotic fraction. Both C2-ceramide and TNFalpha + CHX increased caspase 8- and 3-like activities in cytosolic extracts; however, treatment of cells with the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected NB16 cells from TNFalpha + CHX-induced cell death but did not prevent C2-ceramide cytotoxicity. Although C2-ceramide triggered apoptosis in a fraction of the cells, cell death in the population was primarily caused by necrosis. Thus, C2-ceramide does not faithfully mimic the effects of apoptotic ligands such as TNFalpha, which are thought to be mediated by an accumulation of endogenous ceramide. The inhibition of phosphatidylcholine synthesis is a target for C2-ceramide-mediated cytotoxicity, and this work suggests that other agents that kill cells by inhibiting this pathway may also use a mixture of mechanisms, including necrosis as well as apoptosis.     

Induction of apoptosis in HT-29 colon cancer cells by crude saponin from Platycodi Radix

This study examined the apoptotic effects of crude saponins acquired from the roots of Platycodon grandiflorum (SPR) in HT-29 human colon cancer cells.SPR decreased HT-29 cell proliferation in dose- and time-dependent manners by inducing apoptosis via DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage. The apoptosis induced by SPR was associated with the activation of initiator caspases-8 and -9, as well as the effector caspase-3. SPR stimulated Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. SPR increased the expression of the pro-apoptotic protein, Bax, and decreased the expression of the anti-apoptotic protein, Bcl-2. SPR also increased the expression of the caspase-independent mitochondrial apoptosis factor, AIF, in HT-29 cells. These results indicate that SPR inhibits HT-29 cell proliferation by inducing apoptosis, which may be mediated via both caspase-dependent and -independent pathways.     

Mechanisms of thiosulfinates from Allium tuberosum L.-induced apoptosis in HT-29 human colon cancer cells

This study was performed to elucidate the apoptotic pathways by thiosulfinates, major biologically active components of Allium tuberosum L., in HT-29 human colon cancer cells. Thiosulfinates significantly induced cell death in dose- and time-dependent manners in HT-29 cells, which is associated with apoptosis. Thiosulfinates activated the initiator caspase-8, and -9, and the effector caspase-3. In the present study, thiosulfinates were found to stimulate Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. Thiosulfinates down-regulated the expression of the anti-apoptotic protein Bcl-2, and up-regulated the expression of the pro-apoptotic protein Bax. We also found that thiosulfinates increased the expression of AIF, a caspase-independent mitochondrial apoptosis factor, and induced DNA fragmentation and chromatin condensation in HT-29 cells. These results indicate that thiosulfinates from A. tuberosum L. inhibited cell proliferation and activated both the caspase-dependent and caspase-independent apoptotic pathways in HT-29 cells.     

Role of hesperetin (a natural flavonoid) and its analogue on apoptosis in HT-29 human colon adenocarcinoma cell line--a comparative study

Colon cancer is one of the serious health problems in most developed countries and its incidence rate is increasing in India. Hesperetin (HN) (3',5,7-trihydroxy-4'-methoxyflavonone) and hesperetin analogue (HA) were tested for their apoptosis inducing ability. Methyl thiazolyl tetrazolium assay revealed a dose as well as duration-dependent reduction of HT-29 (colon adenocarcinoma) cellular growth in response to HN and HA treatment. At 24 h 70 μM of HN and 32 μM of HA showed 50% reduction of HT-29 cellular growth. Acridine orange/ethidium bromide staining showed apoptotic features of cell death induced by HN and HA. Rhodamine 123 staining showed significant reduction in mitochondrial membrane potential induced by HN and HA. HN and HA induced DNA damage was confirmed by comet tail formation. Lipid peroxidation markers (TBARS) and protein oxidation marker (PCC) were significantly elevated in HN and HA treated groups. Enzymic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) were slightly decreased in their activities compared to control (untreated HT-29 cells). Results of Western blot analysis of apoptosis associated genes revealed an increase in cytochrome C, Bax, cleaved caspase-3 expression and a decrease in Bcl-2 expression. These findings indicate that HN and HA induce apoptosis on HT-29 via Bax dependent mitochondrial pathway involving oxidant/antioxidant imbalance.     

Anti-proliferative effects of Lethariella zahlbruckneri extracts in human HT-29 human colon cancer cells

This study was performed to elucidate the anti-proliferative effects and the apoptotic mechanisms of extracts from Lethariella zahlbruckneri in HT-29 human colon cancer cells. Both the acetone extract (AEL) and methanolic extract (MEL) of L. zahlbruckneri decreased viable cell numbers in a dose- and time-dependent manner in HT-29 cells. The AEL showed stronger cytotoxicity than MEL. Cell death induced by AEL increased cell populations in the sub-G1 phase, as well as the formation of apoptotic bodies and nuclear condensation, whereas MEL did not. Therefore, the potential of AEL to induce apoptosis was examined. Apoptosis induced by AEL was associated with the activation of initiator caspases-8 and -9, as well as the effector caspase-3. AEL stimulated Bid cleavage. This indicated that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. AEL increased the expression of the pro-apoptotic protein, Bax, and decreased the expression of the anti-apoptotic protein, Bcl-2. AEL also increased the expression of the caspase-independent mitochondrial apoptosis factor, AIF, in HT-29 cells. These results indicate that AEL inhibited HT-29 cell proliferation by inducing apoptosis, which might be mediated via both caspase-dependent and -independent pathways.     

Lipid microsphere preparation of a lipophilic ceramide derivative suppresses colony formation in a murine experimental pulmonary metastasis model

Ceramide is a well-known regulator of apoptosis and cell growth. In this study, we synthesized lipophilic ceramide derivatives to incorporate into lipid microspheres (LM) and their activity was evaluated in vivo. Cera 03, a lipophilic ceramide derivative synthesized from membrane-permeable C2-ceramide, caused potent growth inhibition and DNA fragmentation of Meth A-T tumor cells in vitro. Its potency was similar to that of C2-ceramide. Both compounds increased the proportion of apoptotic cells. Cera 02, the diacetylated form of natural ceramide (Cer), also suppressed in vitro cell growth with a similar or higher potency to that of Cer, but both were far less potent than C2-ceramide and Cera 03. LM containing Cera 03 (Lipo-Cera 03) could not totally prevent metastatic incidence of Meth A-T cells, but reduced pulmonary metastatic nodules in number. Intravenous injection of Lipo-Cera 03 (1 mg/kg of Cera 03) produced about 35% inhibition, while Lipo-Cera 02 had no significant effect. In conclusion, Lipo-Cera 03 may have potential as an antimetastatic drug and may also be a useful tool for researching the role of ceramides in vivo.     

Metabolism of short-chain ceramide by human cancer cells—Implications for therapeutic approaches

Due to recent use of short-chain ceramides in preclinical studies, we characterized C6-ceramide metabolism in cancer cell lines and assessed metabolic junctures for enhancing efficacy. MDA-MB-231 breast cancer cells decreased the amount of C6-ceramide metabolized to C6-sphingomyelin (C6-SM) and increased the amount metabolized to C6-glucosylceramide (C6-GC) in response to increasing concentrations. A similar trend was seen in DU-145 (prostate cancer), PANC-1 (pancreatic cancer), and LoVo (colorectal cancer) cells. KG-1 leukemia cells favored C6-SM synthesis at low (0.6 μM) and high-dose (12 μM) C6-ceramide. Partnering C6-ceramide with tamoxifen, a P-glycoprotein antagonist that impedes ceramide glycosylation, was an effective regimen for enhancing cytotoxicity in cells. Experiments to assess the mechanism of cell death using KG-1 cells showed that tamoxifen inhibited synthesis of C6-GC and C6-SM from C6-ceramide by 80% and 50%, respectively, which was accompanied by enhanced apoptosis. Radiolabeling of KG-1 cells with [³H]palmitic acid produced a 2-fold increase in ³H-long-chain ceramides when unlabeled C6-ceramide was added and a 9-fold increase when C6-ceramide and tamoxifen were added. The increase in ³H-palmitate radiolabeling of long-chain ceramides was blocked by inclusion of a ceramide synthase inhibitor; however, inhibiting synthesis of long-chain ceramide did not rescue cells. These studies show that tamoxifen enhances the apoptotic effects of C6-ceramide. The proposed mechanism involves blocking short-chain ceramide anabolism to favor hydrolysis and generation of sphingosine. We propose that use of tamoxifen and other P-glycoprotein antagonists can be an effective means for enhancing cytotoxic potential of short-chain ceramides in the treatment of cancer.     

Changes in ceramide and sphingomyelin following fludarabine treatment of human chronic B-cell leukemia cells

Fludarabine is used to treat chronic lymphocytic leukemia. Both in vitro and in vivo studies have indicated that apoptosis is an important mode of fludarabine-induced cell death. However, the apoptotic pathways activated are not known. The effects of apoptotic doses of fludarabine on sphingomyelin, ceramide and the production of reactive oxygen species were investigated in the chronic B-cell leukemia lines WSU and JVM-2. Apoptosis, as assessed by an increase in phosphatidylserine externalization, internucleosomal DNA fragmentation and caspase-3-like activity, was evident by 18 h after fludarabine in both cell lines. The general caspase inhibitor t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone (OMe, methyl ester) significantly inhibited apoptosis supporting a role for caspases in fludarabine-induced cell death. A 2.5- to threefold elevation in ceramide levels was observed 6 h after fludarabine treatment. Concomitantly, a decrease in sphingomyelin levels was observed. Fumonisin B1 (an inhibitor of ceramide synthase) pretreatment significantly prevented fludarabine-induced ceramide generation and apoptosis. Conversely, C6-ceramide induced apoptosis in both cell lines. No effect of fludarabine on indices of oxidative stress (dichlorofluorescin oxidation and glutathione disulfide formation) were detected, although partial protection from apoptosis, and prevention of ceramide generation and caspase-3 activation, were achieved with N-acetylcysteine. These findings are consistent with the involvement of caspases and ceramide in fludarabine-induced apoptosis in WSU and JVM-2 cells. Oxidative stress does not appear to be induced by fludarabine, although the protective effects of N-acetylcysteine suggest that thiol redox balance may play a role in the apoptotic pathway.     

A glycoprotein from Laminaria japonica induces apoptosis in HT-29 colon cancer cells

We isolated a novel glycoprotein from the brown alga Laminaria japonica that has antiproliferative effects on HT-29 colon cancer cells. We also identified the mechanism by which this glycoprotein, named LJGP, induces apoptosis. MTS assays showed that LJGP inhibited the proliferation of several cancer cell lines (AGS, HepG2, HT-29) in a dose-dependent manner. Especially in HT-29 cells, proliferation was significantly decreased. LJGP treatment on HT-29 displayed several apoptotic features, such as DNA fragmentation, sub-G1 arrest, caspase-3 activation, and PARP degradation. Consistent with sub-G1 arrest, LJGP decreased the expression of Cdk2, cyclin E, cyclin D1, PCNA, E2F-1, and phosphorylated pRb. Furthermore, the increase of p27 expression was observed. We also determined that LJGP-induced apoptosis leads to the formation of a death-induced signaling complex of Fas, FADD, and procaspase-8. LJGP induced the reduction of mitochondrial membrane potential with activation of the Bcl-2 family of proteins and caspase-9. These findings suggest that LJGP inhibits HT-29 cell proliferation by inducing apoptosis, which may be mediated via multiple pathways, including the Fas signaling pathway, the mitochondrial pathway, and cell cycle arrest. Therefore, LJGP can be a useful treatment option for colon cancer in humans.