Curcumin induces apoptosis involving bax/bcl-2 in human hepatoma SMMC-7721 cells

Curcumin is a major active component of Curcuma aromatica salisb, which has been shown to inhibit proliferation of a wide variety of tumor cells. In this study, the molecular mechanisms of curcumin inducing apoptosis in human hepatoma SMMC-7721 cells were examined. We find that curcumin inhibits the growth of SMMC-7721 cells significantly in a concentration-depenent manner, with typical apoptotic morphological changes of cellular nuclei. Annexin-V/PI double staining detected by flow cytometry and expression of the relative apoptotic proteins (Bax, Bcl-2 and caspase-3) revealed a strong apoptosis-inducing competent of curcumin in SMMC-7721 cells. Curcumin increased the expression of bax protein while decreasing that of bc1-2 protein significantly. The results suggest that curcumin induction of apoptosis involves modulation of bax/bcl-2 in SMMC-7721 cells and provide a molecular basis for the development of naturally compounds as novel anticancer agents for human hepatomas.     

丹参多酚酸盐通过线粒体途径诱导人肝癌SMMC-7721细胞的凋亡

目的：研究丹参多酚酸盐(salvianolate)体外诱导人肝癌细胞SMMC-7721凋亡作用及其可能机制。方法：不同质量浓度丹参多酚酸盐(0.5、1、2 mg/ml)与肝癌细胞共培养24 h后,流式细胞仪检测肝癌细胞凋亡,线粒体膜电位试剂盒(JC-1)检测线粒体膜电位变化;比色法测定1.0 mg/ml丹参多酚酸盐作用后肝癌细胞内caspase8、caspase9 及caspase3的活性,流式细胞仪检测培养体系内加入caspase9抑制剂(zLEHDfmk)或caspase3抑制剂(zDEVDfmk)后细胞凋亡率的变化,Western blotting检测肝癌细胞内线粒体凋亡途径相关蛋白Bax、Bcl2表达水平。结果：丹参多酚酸盐显著诱导肝癌细胞SMMC7721凋亡(P<0.05),同时线粒体膜电位随着药物浓度的升高而加剧下降(P<0.05)。1.0 mg/ml 丹参多酚酸盐处理肝癌细胞24 h后caspase-9与caspase-3的活性明显升高(P<0.05),而caspase-8的活性无明显变化(P>0.05);当培养体系内加入caspase-9或caspase3活性抑制剂后,丹参多酚酸盐诱导肿瘤细胞凋亡的作用明显降低(P<0.05)。Western blotting检测显示,丹参多酚酸盐处理组前凋亡蛋白Bax表达明显升高,抗凋亡蛋白Bcl2表达降低。结论：丹参多酚酸盐(0.5~2.0 mg/ml)剂量具有促进肝癌细胞凋亡的作用,且有剂量依赖的趋势,其机制与线粒体凋亡途径有关。     

沙利度胺对人肝癌细胞株SMMC-7721体外生长的抑制作用

目的 研究沙利度胺对人肝癌细胞株SMMC-7721体外生长的抑制作用及其可能的机制.方法 将不同浓度的沙利度胺作用于人肝癌细胞株SMMC-7721,采用四甲摹偶氮唑蓝(MTT)法检测沙利度胺对SMMC-7721细胞的增殖抑制作用.将SMMC-7721细胞培养至对数生长期,采用DNA琼脂糖凝胶电泳、荧光显微镜观察、流式细胞仪检测等方法 观察沙利度胺处理后SMMC-7721细胞的凋亡梯度、形态学变化和凋亡率,并对凋亡调控蛋白caspase-3的表达进行测定.采用酶联免疫吸附(ELISA)法测定不同浓度的沙利度胺处理后SMMC-7721细胞表达血管内皮生长因子(VEGF)的变化.结果 沙利度胺的浓度从3.125μg/ml增至200μg/ml时,其对SMMC-7721细胞的增殖抑制率从11.7%增至34.2%;当沙利度胺的浓度>25 μg/ml时,其对SMMC-7721细胞的增殖抑制作用明显强于空白对照组(P<0.05).200 μg/ml的沙利度胺处理SMMC-7721细胞24 h后,行琼脂糖凝胶电泳,可见到DNA梯形条带;48 h后梯形条带更明显,并且在荧光显微镜下可见SMMC-7721细胞出现核固缩和核裂解现象.200μg/ml的沙利度胺处理SMMC-7721细胞12、24、48和72 h时,碘化丙啶(PI)法检测SMMC-7721细胞的凋亡率分别为3.1%±0.5%、8.4%±1.3%、19.4%±3.5%和25.8%±2.1%,24 h起的凋亡率均明显高于空白对照组SMMC-7721细胞48 h的自然凋亡率(1.6%±0.6%,均P<0.05).50、100和200μg/ml的沙利度胺处理SMMC-7721细胞48 h时,Annexin V-FITC/PI双标法检测SMMC-7721细胞的凋亡率分别为8.7%±1.2%、16.8%±2.5%和25.4%±4.5%,均明显高于空白对照组SMMC-7721细胞48 h的自然凋亡率(2.1%±0.5%,均P<0.05).随着沙利度胺浓度的增加,表达caspase-3蛋白的SMMC-7721细胞数量不断增加,而SMMC-7721细胞中VEGF的含量却逐渐下降.结论 沙利度胺可能通过诱导肝癌细胞的凋亡、抑制肿瘤血管的生成而发挥双重抗肿瘤生长的作用.     

Synthetic chenodeoxycholic acid derivative, HS-1200, induces apoptosis of human hepatoma cells via a mitochondrial pathway

We investigated whether HS-1200 has anti-proliferation effects on human hepatoma cells in vitro. Here, chromatin condensation, DNA ladder formation and proteolytic cleavage of poly (ADP-ribose) polymerase (PARP) were observed after treatment of HS-1200, indicating the occurrence of apoptotic cell death, which was associated with up-regulation of Bax, cleaved-caspase-3 and cleaved-caspase-9. Inhibition of caspase-9 rescued HS-1200-induced apoptosis. Furthermore, cells treated with HS-1200 showed a reduction in mitochondrial membrane potential (Δψ_m) and caused cytochrome c release into the cytosol. The results indicated that synthetic chenodeoxycholic acid HS-1200 could induce cell apoptosis in BEL7402 human hepatoma cell line, via a Bax/cytochrome c/caspase-9 independent pathway. This study suggested that HS-1200 is potentially useful as an apoptosis inducer for the treatment of hepatocellular carcinoma.     

积雪草酸对人肝癌SMMC-7721细胞增殖和自噬的影响

目的  探讨积雪草酸（asiatic acid,AA）对人肝癌SMMC-7721细胞增殖和自噬的影响。方法 不同浓度AA作用于人肝癌SMMC-7721细胞24 h后,MTT法检测细胞活性并观察自噬抑制剂3-MA对40 μmol/L AA的干预作用;MDC染色检测自噬泡的形成,Western blot检测自噬相关蛋白LC3-Ⅰ、LC3-Ⅱ、p62、mTOR、p-mTOR及p53的表达。结果 MTT检测结果显示,不同AA浓度均可抑制人肝癌SMMC-7721细胞增殖,并呈浓度依赖性（F＝46.790,P＝0.006）,IC₅₀ =37.313 μmol/L。与单独使用40 μmol/L AA相比,自噬抑制剂3-MA可部分逆转40 μmol/L AA对SMMC-7721细胞增殖的抑制作用［（46.400±9.099）% vs （22.000±3.391）%,P＜0.001］。MDC染色实验表明40 μmol/L AA干预可增加自噬泡形成。Western blot检测发现,与对照组比较,40 μmol/L AA可明显降低LC3-Ⅰ的蛋白表达,而提高LC3-Ⅱ表达（1.744±0.108 vs 1.529±0.065,t=2.928,P=0.043;0.113±0.031 vs 0.380±0.036,t=-9.754,P<0.001）,降低p62蛋白表达（0.522±0.024 vs 0.123±0.019,t=22.565,P<0.001）和p-mTOR蛋白表达（1.252±0.039 vs 0.353±0.028,t=30.775,P<0.001）,但对mTOR和p53的蛋白表达无影响（1.713±0.111 vs 1.556±0.076,t=1.555,P=0.190;0.671±0.040 vs 0.726±0.055,t=-1.555,P=0.210）。结论 AA能抑制人肝癌SMMC-7721细胞增殖,可能与其通过非p53依赖方式负调控mTOR通路诱发自噬有关。     

粉防己碱诱导人视网膜母细胞瘤细胞凋亡及其机制研究

背景与目的：粉防己碱(tetrandrine,Tet)是一种天然化合物,其抗视网膜母细胞瘤作用尚不清楚。该研究拟检测Tet对人视网膜母细胞瘤细胞的抗肿瘤作用,并进一步阐明其作用机制。方法：采用CCK-8法检测Tet对视网膜母细胞瘤细胞活力的抑制作用;应用Annexin V/PI法检测细胞凋亡情况;在2’,7’-二氯荧光素二乙酸酯(2’, 7’-dichlorofluorescin diacetate,DCFH-DA)染色后,采用流式细胞术检测细胞内反应性活性氧(reactive oxygen species,ROS)含量;采用蛋白［质］印迹法(Western blot)检测细胞Akt、p-Akt蛋白表达量。结果：Tet显著抑制视网膜母细胞瘤细胞活力,Tet浓度为4、8、10和20 μmol/L处理细胞24 h时,WERI-Rb-1细胞抑制率分别为5.7%、25.0%、55.1%和84.9%,Y79细胞抑制率分别为2.4%、2.9%、23.8%和54.2% (P<0.01);10 μmol/L Tet处理细胞12、24和48 h时,WERI-Rb-1细胞抑制率分别为6.0%、45.5%和74.7%,Y79细胞抑制率分别为2.9%、19.4%和43.3%(P<0.01)。Tet诱导细胞凋亡,以10 μmol/L Tet处理细胞24和48 h时,WERI-Rb-1细胞凋亡率分别为(23.70±1.75)%和(34.83±3.15)%,Y79细胞凋亡率分别为(9.62±2.69)%和(14.97±1.50)%(P<0.01),凋亡抑制剂Z-VAD-FMK能够显著抑制Tet对视网膜母细胞瘤细胞的凋亡诱导作用(P<0.05)。10 μmol/L Tet作用细胞6及12 h后,细胞的ROS产生量较对照组明显上升(P<0.01),N-乙酰基-L-半胱氨酸(N-acetyl-L-cysteine,NAC)能够抑制Tet诱导产生的ROS(P<0.01),NAC抑制ROS后,细胞的凋亡率较单独Tet作用组明显下降(P<0.01)。Tet能够抑制视网膜母细胞瘤细胞PI3K/Akt信号通路。结论：Tet诱导视网膜母细胞瘤细胞凋亡,该作用机制与细胞内ROS升高、PI3K/Akt信号通路抑制有关。     

异硫氰酸苯己酯调控肝癌细胞株SMMC-7721组蛋白乙酰化及诱导细胞凋亡

目的 研究异硫氰酸苯己酯(PHI)对肝癌SMMC-7721细胞组蛋白乙酰化调控及凋亡的影响.方法 采用台盼蓝拒染直接计数法观察PHI对SMMC-7721细胞增殖的影响,采用原位末端标记(TUNEL)法检测PHI对SMMC-7721细胞凋亡的影响,采用Western blot法检测PHI对SMMC-7721细胞组蛋白乙酰化及凋亡相关蛋白表达的影响.结果 PHI可抑制SMMC-7721细胞的增殖,与0 μmol/L作用组比较,5、10、20、40和80 μmol/L的PHI对SMMC-7721细胞均有不同程度的增殖抑制作用.PHI可诱导SMMC-7721细胞产生凋亡,PHI作用于SMMC-7721细胞7 h后,10、20和40 μmol/LPHI组的细胞凋亡率分别为6.9%±2.4%、17.5%±4.2%和54.5%±5.4%,明显高于0 μmol/L PHI组(4.5%±2.3%,P＜0.05).PHI作用于SMMC-7721细胞3 h时,与0 μmol/L PHI组比较,10、20和40 μmol/L PHI组中Bcl-2、Procaspse-9和Procaspse-3的表达下降,caspase-9和caspase-3的表达上升,而Procaspase-8的表达未见明显变化;作用7 h时,这种变化趋势更加明显.PHI作用于SMMC-7721细胞3 h时,与0 μmol/L PHI组比较,10、20和40 μmol/L PHI组中组蛋白H3的乙酰化分别增加了1.87倍、2.43倍和3.67倍,组蛋白H4的乙酰化分别增加了1.29倍、1.45倍和2.25倍;作用7 h时,这种变化趋势更加明显.结论 PHI是一种组蛋白去乙酰化酶抑制剂,可调控组蛋白的乙酰化水平,影响其表观遗传学,并通过线粒体凋亡途径诱导细胞凋亡.     

A new jasmonic acid stereoisomeric derivative induces apoptosis via reactive oxygen species in human prostate cancer cells

With the aim of identifying novel agents with antigrowth and pro-apoptotic activity on prostate cancer cells, in the present study, we evaluated the effect of a (−)-jasmonic acid derivative, the 3-hydroxy-2(S)-(2Z-butenyl)-cyclopentane-1(S)-acetic acid, obtained by biotransformation, on cell growth in androgen-sensitive (LNCaP) and androgen-insensitive (DU-145) human prostate cancer cells. The results obtained show that the new compound was able to inhibit the growth of both prostate cancer cells. In addition, our data seem to indicate that the apoptosis evocated by this new molecule, at least in part, appears to be associated with an increase of reactive oxygen species (ROS) production.     

Iso-suillin Isolated from Suillus luteus,Induces G1 Phase Arrest and Apoptosis in Human Hepatoma SMMC-7721 Cells

Iso-suillin, a natural product isolated from Suillus luteus, has been shown to inhibit the growth of somecancer cell lines. However, the molecular mechanisms of action of this compound are poorly understood. Thepurpose of this study was to investigate how iso-suillin inhibits proliferation and induces apoptosis in a humanhepatoma cell line (SMMC-7721). We demonstrated the effects of iso-suillin on cell proliferation and apoptosisin SMMC-7721 cells, with no apparent toxicity in normal human lymphocytes, using colony formation assaysand 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Western blotting was usedto examine the expression of G1 phase-regulated and apoptosis-associated protein levels in iso-suillin treatedSMMC-7721 cells. The results indicated that iso-suillin significantly decreased viability, induced G1 phase arrestand triggered apoptosis in SMMC-7721cells. Taken together, these results suggest the potential of iso-suillin asa candidate for liver cancer treatment.     

Simvastatin inhibition of mevalonate pathway induces apoptosis in human breast cancer cells via activation of JNK/CHOP/DR5 signaling pathway

Simvastatin (SVA) was shown to up-regulate expression of death receptor-5 (DR5), CCAAT/enhancer binding protein homologous protein (CHOP) and phosphorylated c-Jun N-terminal kinase (pJNK) in human breast cancer cell lines. siRNA knockdown of DR5, CHOP or JNK significantly blocked SVA-induced apoptosis, demonstrating the importance of JNK/CHOP/DR5 signaling pathway in SVA-induced apoptosis. Exogenous addition of either mevalonate or geranylgeranyl pyrophosphate (GGPP) inhibited SVA activation of JNK/CHOP/DR5 pro-apoptotic pathway, indicating that activation of JNK/CHOP/DR5 pro-apoptotic pathway is dependent on SVA inhibition of 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase and its intermediate GGPP. Data provide novel insight into better understanding the anticancer mechanisms of SVA.     

过表达PRRX1 通过p53 介导的线粒体凋亡途径诱导肝癌SMMC7721 细胞凋亡

[摘要] 目的：探讨配对相关同源框1 蛋白（PRRX1）过表达对肝癌SMMC7721 细胞凋亡的影响及其分子机制。方法：分别用慢病毒介导PRRX1 过表达载体（pGMLV-PRRX1）、空载质粒（Vector）感染人肝癌SMMC7721 细胞,用qPCR和WB实验检测慢病毒感染后细胞中PRRX1 mRNA和蛋白的表达变化,用CCK-8 法、Annexin-V FITC/PI 染色流式细胞术分别检测PRRX1 过表达对SMMC7721 细胞增殖、凋亡的影响,用线粒体膜电位检测试剂盒（JC-10 染色法）检测细胞线粒体膜电位变化,用caspase 活性检测试剂盒（分光光度法）测定细胞中caspase-8 和caspase-9 酶活性,用WB实验检测细胞中p53、Bcl-2、Bax、Fas、Cleaved-caspase-3以及线粒体和细胞质中细胞色素C（Cty C）蛋白的表达。结果：成功构建PRRX1 过表达的SMMC7721 细胞株,感染细胞中PRRX1 mRNA和蛋白的表达水平显著升高（均P<0.01）。与对照组和空载组比较,PRRX1 过表达组SMMC7721 细胞的增殖能力显著下降、细胞凋亡率显著增高、Cleaved-caspase-3 剪切水平显著升高、线粒体膜电位显著下降、线粒体中Cty C蛋白表达下调、胞质中Cty C蛋白表达上调以及caspase-9 酶活性升高（P<0.05 或P<0.01）,同时p53 和Bax 蛋白表达增加而Bcl-2 蛋白表达降低（均P<0.05）,但Fas 蛋白表达及caspase-8 酶活性无显著变化（均P>0.05）。结论: PRRX1 过表达可诱导肝癌SMMC7721 细胞凋亡,其机制可能与p53介导的线粒体凋亡途径被激活有关。     

珠子参体外诱导人肝癌细胞凋亡效应及机制研究

目的观察珠子参体外诱导人肝癌细胞凋亡效应并初探其分子机制。方法体外细胞培养采用人肝癌细胞株SMMC-7721,分为对照(BL)组、珠子参(PJ)组、二甲基亚砜(DMSO)组及5-FU组,采用电镜观察作用后肝癌细胞超微结构改变;流式细胞仪检测肝癌细胞周期和凋亡率;RT-PCR法检测癌基因c-myc、c-fos和抑癌基因p53、p21表达的变化。结果与对照组比较,电镜下珠子参组SMMC-7721细胞染色质浓缩,分解成大小不一有膜包绕团块,内含有新月形DNA物质及细胞器,形成凋亡小体;周期分析可见G0/G1期细胞阻滞,阻止了细胞向S期的转换,并引起细胞凋亡,凋亡率达38.34%;RT-PCR半定量分析珠子参能降低癌基因c-myc表达(P<0.05),增高抑癌基因p53和p21表达(P<0.05)。结论珠子参能诱导人肝癌细胞SMMC-7721凋亡,部分作用机制可能与阻滞细胞停留在G0/G1,降低癌基因c-myc和c-fos表达,增高抑癌基因p53和p21表达有关。     

Andrographolide enhances 5-fluorouracil-induced apoptosis via caspase-8-dependent mitochondrial pathway involving p53 participation in hepatocellular carcinoma (SMMC-7721) cells

Despite recent significant advances in the treatment of human carcinoma (HCC), the results of chemotherapy to date remain unsatisfactory. 5-Fluorouracil (5-FU) still represents the cornerstone of treatment of carcinoma, and resistance to the actions of 5-FU is a major obstacle to successful chemotherapy. More effective treatment strategies may involve combinations of agents with activity against HCC. Andrographolide (ANDRO), a natural bicyclic diterpenoid lactone isolated from Andrographis paniculata, has been shown to suppress the growth of HCC cells and trigger apoptosis in vitro. To assess the suitability of ANDRO as a chemotherapeutic agent in HCC, its cytotoxic effects have been evaluated both as a single agent and in combination with 5-FU. ANDRO potentiates the cytotoxic effect of 5-FU in HCC cell line SMMC-7721 through apoptosis. ANDRO alone induces SMMC-7721 apoptosis with p53 expression, Bax conformation and caspase-3,8,9 activation. Surprisingly, the addition of ANDRO to 5-FU induces synergistic apoptosis, which could be corroborated to the increased caspase-8, p53 activity and the significant changes of Bax conformation in these cells, resulting in increased losses of mitochondrial membrane potential, increased release of cytochrome c, and activation of caspase-9 and caspase-3. Suppression of caspase-8 with the specific inhibitor z-IETD-fmk abrogates largely ANDRO/5-FU biological activity by preventing mitochondrial membrane potential disappearance, caspase-3,9 activation and subsequent apoptosis. The results suggest that ANDRO may be effective in combination with 5-FU for the treatment of HCC cells SMMC-7721.     

Jaridonin耗竭谷胱甘肽诱导DNA损伤致食管癌细胞凋亡

目的 探讨Jaridonin诱导食管癌细胞凋亡的作用机制.方法 采用单细胞凝胶电泳法检测DNA损伤.Western blot法检测p53蛋白的表达.谷胱甘肽(GSH)检测试剂盒检测细胞内还原性GSH水平.采用氧化还原敏感探针2’,7'-二氯荧光素二乙酸酯和二氢乙锭染色,荧光显微镜和流式细胞仪分别检测细胞内过氧化氢(H2O2)和超氧阴离子(O2.-)水平.结果 20、40 μmol/L Jaridonin 处理组的Olive尾矩值分别为45.2 ±8.1和89.0±14.2,与对照组(3.2±2.3)比较,差异均有统计学意义(均P＜0.01).Jaridonin处理EC-1细胞2h时,p53表达开始上调,且随作用时间延长,p53表达水平升高.siRNA干扰p53表达后,EC-1细胞对Jaridonin表现出明显的抗性,细胞凋亡率由转染前的38.5％下降为转染后的8.8％.Jaridonin作用EC-1细胞后,H2O2水平显著升高,O 2.-水平无明显变化.20 μmol/L Jaridonin作用EC-1细胞前后,GSH水平分别为(10.3 ±1.6)nmol/mg蛋白和(4.6±2.1)nmol/mg蛋白,差异有统计学意义(P＜0.01),随药物浓度的增加,GSH水平进一步下降.抗氧化剂GSH逆转了Jaridonin诱导的EC-1细胞中H2O2水平的升高、DNA损伤和细胞凋亡.Jaridonin对人正常肝细胞L-02的生长情况无明显影响.结论 Jaridonin通过消耗GSH的含量引起H2O2水平的升高而诱导DNA损伤,最终选择性诱导了食管癌细胞的凋亡.     

Involvement of reactive oxygen species in 2-methoxyestradiol-induced apoptosis in human neuroblastoma cells

Neuroblastoma is the most common extra-cranial solid tumor in children. Despite advances in the treatment of childhood cancer, outcomes for children with advanced-stage neuroblastoma remain poor. Here we reported that 2-methoxyestradiol (2-ME) inhibited the proliferation and induced apoptosis in human neuroblastoma SK-N-SH and SH-SY5Y cells. 2-ME treatment also resulted in the generation of ROS and the loss of mitochondrial membrane potential in SK-N-SH and SH-SY5Y, indicating that 2-ME-induced apoptosis is mediated by ROS. This is supported by the results that have shown that co-treatment with antioxidants, VC, L-GSH and MitoQ₁₀, decreased 2-ME-induced generation of ROS and the loss of the mitochondrial membrane potential, increased the Bcl-2/Bax ratio, decreased 2-ME-induced activation of caspase-9 and caspase-3 and the up-regulation of apoptosis-inducing factor (AIF), and prevented 2-ME-induced apoptosis in SK-N-SH and SH-SY5Y cells. These results suggested that oxidative stress plays an important role in 2-ME-induced apoptotic death of human neuroblastoma cells.     

Reactive oxygen species-mediated endoplasmic reticulum stress and mitochondrial dysfunction contribute to cirsimaritin-induced apoptosis in human gallbladder carcinoma GBC-SD cells

In this study, the anticancer effect of cirsimaritin, a natural flavonoid, against human gallbladder carcinoma cell line GBC-SD and the underlying mechanisms were investigated. Cirsimaritin inhibited the growth of tumor cells and induced mitochondrial apoptosis in GBC-SD cells. In addition, cirsimaritin triggered endoplasmic reticulum (ER) stress and down-regulated the phosphorylation of Akt, while knock-down of CHOP dramatically abrogated the inactivation of Akt and reversed the pro-apoptotic effect of cirsimaritin. Furthermore, cirsimaritin provoked the generation of reactive oxygen species in GBC-SD cells, while the antioxidant N-acetyl cysteine almost completely blocked the activation of ER stress and apoptosis, suggesting cirsimaritin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways in GBC-SD cells.     

VI-16, a newly synthesized flavonoid, induces apoptosis through the mitochondrial pathway in human hepatoma cells

VI-16, a newly synthesized flavonoid, has a hydroxy substitution at C5 position, a methoxyl substitution at C5 position, and a piperazine substitution at C7 position. Here, we firstly investigated the potential antitumor effect of VI-16 in HepG2 human hepatocarcinoma cells. The MTT assay showed that VI-16 inhibited HepG2 cell growth in a concentration- and time-dependent manner. To further investigate whether apoptosis induction contributed to the antitumor effects of VI-16, DAPI staining and Annexin-V/PI double staining were performed in our tests. The data showed that VI-16 could induce apoptotic cell death in HepG2 cells. Moreover, mechanistic studies revealed that VI-16-induced apoptosis was a caspase-dependent process by decreasing the expression of pro-caspase-3. The changes in the expression of caspase-8, capsase-9, Bax and bcl-2 after VI-16 treatment suggested that the mitochondrial pathway was involved in the apoptosis induced by VI-16. Furthermore, VI-16 could significantly increase the loss of mitochondrial membrane potential and the expression of p53. Taken together, these results demonstrated that apoptosis induced by VI-16 might be one of the mechanisms by which VI-16 acts as a preventive antitumor drug against human hepatoma.     

Ceramide induces mitochondrial activation and apoptosis via a Bax-dependent pathway in human carcinoma cells

The intracellular pathways leading to mitochondrial activation and subsequent cell death in the ceramide-mediated stress response have been intensively studied in recent years. Experimental evidence has been provided that ceramide-induced apoptosis is inhibited by overexpression of antiapoptotic proteins of the Bcl-2 family. However, the direct effect of proapoptotic gene products, e.g. Bax, on ceramide-induced death signalling has not yet been studied in detail. In the present work, we show by measurement of mitochondrial permeability transition, cytochrome c release, activation of caspase-3 and DNA fragmentation that ceramide-induced apoptosis is marginal in Bax-negative DU 145 cells. Reconstitution of Bax by generation of DU 145 cells stably expressing this proapoptotic factor, clearly enhanced ceramide-induced apoptosis at all levels of the mitochondrial signalling cascade. Using the broad-range caspase inhibitor zVAD-fmk and zDEVD-fmk, an inhibitor of caspase-3-like activities, we demonstrate that the ceramide-induced mitochondrial activation in Bax-transfected DU 145 cells is caspase-independent. On the other hand, apoptotic events located downstream of the mitochondria, e.g. DNA fragmentation, were shown to be caspase-dependent. This influence of Bax on ceramide-induced apoptosis was confirmed in another cellular system: whereas Bax-positive HCT116 wild type cells were very sensitive towards induction of cell death by C(2)-ceramide, sensitivity of Bax knock-out HCT116 cells was significantly reduced. Thus, we conclude that Bax is a key activator of ceramide-mediated death pathways.     

丹参酚酸B调控ROS抑制大肠癌细胞HCT-116增殖并促进其凋亡

目的 验证丹参酚酸B(Salvianolic acid B,SalB)抑制人大肠癌细胞HCT-116增殖并促进其凋亡,进一步通过活性氧簇(Reactive oxygen species,ROS)阐述其可能机制。方法 体外培养大肠癌细胞HCF-116,分成HCT-116组、HCT-116+H₂O₂组、HCT-116+SalB组、HCT-116+SalB+N-乙酰-L-半胱氨酸(N-acetylcysteine,NAC)组。分组干预后,采用MTT法检测细胞存活率、平板克隆实验检测细胞增殖、流式细胞术检测ROS含量、细胞周期及细胞凋亡率。结果 SalB对HCT-116细胞具有抑制作用,且在一定浓度范围内呈正相关(P＜0.01);SalB、H₂O₂促进HCT-116细胞内ROS生成(P＜0.01),ROS清除剂NAC预处理可清除由SalB产生的ROS(P＜0.01);SalB抑制HCT-116细胞增殖(P＜0.01)并促进其凋亡(P＜0.01),该作用可被NAC部分逆转(P＜0.05);SalB引起HCT-116细胞G0/G1周期阻滞(P＜0.01),NAC预处理完全逆转SalB导致的周期阻滞(P＜0.05)。结论 SalB可通过增加HCT-116细胞内ROS水平引起细胞周期阻滞,从而抑制细胞增殖,并促进其凋亡。