Cantharidin induces apoptosis in human bladder cancer TSGH 8301 cells through mitochondria-dependent signal pathways

Cantharidin has shown potent anticancer activities on many types of human cancer cells. This study was performed to elucidate whether mitochondria and caspases are involved in the modulation of apoptosis and cell cycle arrest by cantharidin in human bladder cancer cells. The effect of cantharidin on cell cycle arrest, apoptosis, caspases, reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨ(m)) were measured by flow cytometry, and the levels of apoptosis-associated proteins and its regulatory molecules were studied by Western blotting. Cantharidin-induced apoptosis and DNA damage was determined by flow cytometric analysis, DAPI staining and Comet assay. After cantharidin treatment, the active forms of caspase-3, -8 and -9 were promoted. Cantharidin-induced apoptosis was associated with enhanced ROS and Ca(2+) generations, caused DNA damage, decreased the levels of ΔΨ(m) and promoted Endo G and AIF released from mitochondria. Cantharidin-induced G0/G1 arrest was associated with a marked decrease in the protein expressions of cyclin E and Cdc25c but promoted the levels of p21 and p-p53. Cantharidin-induced apoptosis was accompanied with up-regulation of the protein expression of Bax and PARP, but down-regulation of the protein levels of Bcl-2, resulting in dysfunction of mitochondria then led to Endo G and AIF release for causing induction of apoptosis.     

Mitotic catastrophe constitutes a special case of apoptosis whose suppression entails aneuploidy

A conflict in cell cycle progression or DNA damage can lead to mitotic catastrophe when the DNA structure checkpoints are inactivated, for instance when the checkpoint kinase Chk2 is inhibited. Here we show that in such conditions, cells die during the metaphase of the cell cycle, as a result of caspase activation and subsequent mitochondrial damage. Molecular ordering of these phenomena reveals that mitotic catastrophe occurs in a p53-independent manner and involves a primary activation of caspase-2, upstream of cytochrome c release, followed by caspase-3 activation and chromatin condensation. Suppression of caspase-2 by RNA interference or pseudosubstrate inhibitors as well as blockade of the mitochondrial membrane permeabilization prevent the mitotic catastrophe and allow cells to further proceed the cell cycle beyond the metaphase, leading to asymmetric cell division. Heterokarya generated by the fusion of nonsynchronized cells can be driven to divide into three or more daughter cells when Chk2 and caspases are simultaneously inhibited. Such multipolar divisions, resulting from suppressed mitotic catastrophe, lead to the asymmetric distribution of cytoplasm (anisocytosis), DNA (anisokaryosis) and chromosomes (aneuploidy). Similarly, in a model of DNA damage-induced mitotic catastrophe, suppression of apoptosis leads to the generation of aneuploid cells. Our findings delineate a molecular pathway through which DNA damage, failure to arrest the cell cycle and inhibition of apoptosis can favor the occurrence of cytogenetic abnormalities that are likely to participate in oncogenesis.     

非小细胞肺癌细胞中OGG1调控顺铂诱导的细胞毒性的作用机制探讨

目的:探讨8-氧鸟嘌呤DNA糖基化酶(OGG1)在人肺癌细胞中调控顺铂诱导的细胞毒性的作用机制。方法:采用酶联免疫吸附试验、免疫印迹、流式细胞术和免疫荧光法、线粒体拷贝数和膜电位检测等方法,进行相关实验。结果:顺铂可诱导8-羟基-2'-脱氧鸟苷（8-OHdG）浓度升高,8-OHdG的浓度及细胞凋亡分别与OGG1 mRNA的表达存在显著相关性。外源性表达OGG1促进其DNA修复活性,可降低顺铂诱导的8-OHdG水平,提高非小细胞肺癌（non-small cell lung cancer,NSCLC）细胞活力,并抑制顺铂诱导的NSCLC细胞凋亡,而OGG1沉默则表现相反的结果。此外,过表达OGG1减少顺铂导致的线粒体DNA损伤和功能障碍,沉默OGG1增强顺铂诱导的MAPK/p38通路的激活,导致NSCLC细胞发生凋亡。结论:OGG1的下调有利于提高肺癌细胞对顺铂的敏感性。因此,本研究通过在体外证实OGG1介导的DNA修复在顺铂耐药中的作用,为肺癌的治疗提供了理论支持。     

Puma基因转染对胃癌SGC-7901细胞的促凋亡作用及机制

[目的]探讨puma基因转染对人胃癌SGC-7901细胞株增殖和凋亡的影响及其作用机制。[方法]应用脂质体介导重组真核表达载体pEGFP-C1-PUMA瞬时转染至SGC-7901细胞。分别用荧光显微镜和RT—PCR法检测外源基因的表达，MTF比色法测定细胞增殖的抑制，Hoechst33342染色法检测细胞凋亡，流式细胞仪检测细胞周期和线粒体膜电位的变化，Westernblot检测细胞色素C（CytC）和凋亡诱导因子（AIF）的转位。[结果]外源性puma基因在pEGFP—C1-PUMA转染的SGC-7901细胞中实现了表达。PUMA表达使SGC-7901细胞的增殖能力降低并诱导凋亡，转染24、48、72h的生长抑制率分别为19．3％、34．7％、42．2％，凋亡率分别为19．6％、35．4％、46．6％。PUMA表达的SGC-7901细胞DNA合成受到抑制，周期阻滞在GgG1期；线粒体膜电位明显下降，CytC、AIF从线粒体进入胞浆。[结论]puma基因转染可有效抑制胃癌SGC-7901细胞的增殖，促进其凋亡。促凋亡机制主要是线粒体途径，与线粒体膜电位降低和CytC、AIF从线粒体释放有关。     

Antimycin A as a mitochondrial electron transport inhibitor prevents the growth of human lung cancer A549 cells

Antimycin A (AMA) inhibits mitochondrial electron transport between cytochromes b and c. We evaluated the effects of AMA on the growth of human pulmonary adenocarcinoma A549 cells in relation to cell cycle and apoptosis. Treatment with 2-100 microM AMA significantly inhibited the cell growth of A549 for 72 h. DNA flow cytometry indicated that AMA slightly induced a G1 phase arrest of the cell cycle for 72 h. Treatment with 50 microM AMA induced apoptosis of approximately 17% in view of annexin V-staining cells. The dose of 50 microM AMA also induced loss of the mitochondrial membrane potential (DeltaPsi(m)) of approximately 38%. The intracellular reactive oxygen species (ROS) levels including O2(.-) were significantly increased in AMA-treated A549 cells. In conclusion, AMA inhibited the growth of A549 cells via inducing cell cycle arrest as well as triggering apoptosis. Growth inhibition in AMA-treated A549 cells was accompanied by an increase in ROS levels.     

Apoptotic response of HL-60 human leukemia cells to the antitumor drug TAS-103

TAS-103 is a DNA intercalating indeno-quinoline derivative that stimulates DNA cleavage by topoisomerases. This synthetic drug has a broad spectrum of antitumor activity against many human solid tumor xenografts and is currently undergoing clinical trials. We investigated the induction of apoptosis in human promyelocytic leukemia cells treated with TAS-103. The treatment of proliferating human leukemia cells for 24 h with various concentrations of the drug induces significant variations in the mitochondrial transmembrane potential (delta(psi)mt) measured by flow cytometry using the fluorochromes 3,3-dihexyloxacarbocyanine iodide, Mitotracker Red, and tetrachloro-tetraethylbenzimidazolcarbocyanine iodide. The collapse of delta(psi)mt is accompanied by a marked decrease of the intracellular pH. Cleavage experiments with the substrates N-acetyl-Asp-Glu-Val-Asp-pNA, poly(ADP-ribose) polymerase, and pro-caspase-3 reveal unambiguously that caspase-3 is a key mediator of the apoptotic pathway induced by TAS-103. Caspase-8 is also cleaved, and the bcl-2 oncoprotein is underexpressed. Drug-induced internucleosomal DNA fragmentation and the externalization of phosphatidylserine residues in the outer leaflet of the plasma membrane were also characterized. The cell cycle perturbations produced by TAS-103 can be connected with the changes in deltapsi(mt). At low concentrations (2-25 nM), the drug induces a marked G2 arrest and concomitantly provokes an increase in the potential of mitochondrial membranes. In contrast, treatment of the HL-60 cells with higher drug concentrations (50 nM to 1 microM) triggers massive apoptosis and a collapse of deltaP(mt) that is a signature for the opening of the mitochondrial permeability transition pores. The discovery of a correlation between the G2 arrest and changes in mitochondrial membrane potential provides an important mechanistic insight into the action of TAS-103.     

新型多胺缀合物NNAMB诱导K562细胞凋亡及其机制

目的 探讨新型多胺缀合物NNAMB诱导K562细胞凋亡及其分子机制.方法 采用二苯基溴化四氮唑蓝(MTT)法、台盼监拒染法检测细胞活力;Hoechst33258染色观察细胞形态变化;流式细胞仪检测细胞周期变化、凋亡率、线粒体膜电位的变化;Western blot检测caspase-3、caspase-8、caspase-9和cytochrome c的表达.结果 NNAMB抑制K562细胞的生长,并呈现剂量及时间依赖性.随着NNAMB浓度的增加和作用时间的延跃,Sub-G1期细胞明显增加,线粒体膜电位下降.经NNAMB处理后的K562细胞中,可见到明显的凋亡细胞.蛋白印迹检测表明,NNAMB可诱导caspase-3、easpase-9的活化及线粒体cytochrome c释放到胞浆中,但caspase-8的表达无明显变化.结论 NNAMB能显著抑制K562细胞增殖,并可通过内源性线粒体途径诱导细胞凋亡.     

Apoptosis induced by oxaliplatin in human colon cancer HCT15 cell line

BACKGROUND: Oxaliplatin (L-OHP), active in a wide range of human and animal tumours, also CDDP-resistant, possesses unique molecular characteristics of action. However, the mechanisms by which the damage induced by L-OHP triggers a death signal are not yet fully defined. MATERIALS AND METHODS: After L-OHP treatment of the HCT15 human colon cancer cell line, apoptosis was evaluated by DNA laddering detection and by flow cytometry; the effect on specific caspase-3, -8 and -9 inhibitors, mitochondrial membrane permeability transition, cytochrome C release and expression of CD95 and CD95L were also assessed. RESULTS: HCT15 cells underwent apoptosis when treated with all used drug concentrations (7-25 microM). Treatment of cells with L-OHP resulted in the activation of caspase-8, -9 and -3, in a mitochondrial membrane depolarisation, and in an increase of CD95 receptor and CD95 ligand levels. CONCLUSION: The results correlated well with the ability of L-OHP to induce apoptosis and give further insights into the mechanisms underlying the L-OHP-induced apoptosis of tumor cells.     

Anti-hepatoma cells function of luteolin through inducing apoptosis and cell cycle arrest

The aim of this study is to explore the apoptotic induction and cell cycle arrest function of luteolin on the liver cancer cells and the related mechanism. The liver cancer cell line SMMC-7721, BEL-7402, and normal liver cells HL-7702 were treated with different concentrations of luteolin. Cell proliferation ability was tested. Morphological changes of the apoptotic cells were observed under inverted fluorescence microscope after Hoechst33342 staining. We investigated the effect of luteolin on cell cycling and apoptosis with flow cytometry. The mitochondrial membrane potential changes were analyzed after JC-1 staining. Caspases-3 and Bcl-2 family proteins expression were analyzed by real-time PCR. Cell proliferation of SMMC-7721 and BEL-7402 were inhibited by luteolin, and the inhibition was dose–time-dependent. Luteolin could arrest the cells at G1/S stage, reduce mitochondrial membrane potential, and induce higher apoptosis rate and the typical apoptotic morphological changes of the liver carcinoma cells. Q-RT-PCR results also showed that luteolin increased Bax and caspase-3 expression significantly and upregulated Bcl-2 expression in a dose-dependent manner in liver carcinoma cells. However, the normal liver cells HL-7702 was almost not affected by luteolin treatment. Luteolin can inhibit SMMC-7721 and BEL-7402 cell proliferation in a time- and dose-dependent manner. And the mechanism maybe through arresting cell cycle at phase G1/S, enhancing Bax level, reducing anti-apoptotic protein Bcl-2 level, resulting in activating caspase-3 enzyme and decrease of mitochondrial membrane potential, and finally leading to cell apoptosis.     

Targeting human 8-oxoguanine DNA glycosylase (hOGG1) to mitochondria enhances cisplatin cytotoxicity in hepatoma cells

Many chemoradiation therapies cause DNA damage through oxidative stress. An important cellular mechanism that protects cells against oxidative stress involves DNA repair. One of the primary DNA repair mechanisms for oxidative DNA damage is base excision repair (BER). BER involves the tightly coordinated function of four enzymes (glycosylase, apurinic/apyrimidinic endonuclease, polymerase and ligase), in which 8-oxoguanine DNA glycosylase 1 initiates the cycle. An imbalance in the production of any one of these enzymes may result in the generation of more DNA damage and increased cell killing. In this study, we targeted mitochondrial DNA to enhance cancer chemotherapy by over-expressing a human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene in the mitochondria of human hepatoma cells. Increased hOGG1 transgene expression was achieved at RNA, protein and enzyme activity levels. In parallel, we observed enhanced mitochondrial DNA damage, increased mitochondrial respiration rate, increased membrane potential and elevated free radical production. A greater proportion of the hOGG1-over-expressing hepatoma cells experienced apoptosis. Following exposure to a commonly used chemotherapeutic agent, cisplatin, cancer cells over-expressing hOGG1 displayed much shortened long-term survival when compared with control cells. Our results suggest that over-expression of hOGG1 in mitochondria may promote mitochondrial DNA damage by creating an imbalance in the BER pathway and sensitize cancer cells to cisplatin. These findings support further evaluation of hOGG1 over-expression strategies for cancer therapy.     

Coleusin factor exerts cytotoxic activity by inducing G0/G1 cell cycle arrest and apoptosis in human gastric cancer BGC-823 cells

Coleusin factor (CF), a kind of diterpenoids, is isolated and purified from the root of a Chinese tropical plant Coleus forskohlii by our laboratory. Our previous studies have demonstrated that CF significantly inhibits growth in some kinds of cancer cell lines. Here, we found that CF remarkably inhibited growth in human gastric cancer BGC-823 cells by decreasing cell proliferation, inducing G(0)/G(1) cell cycle arrest and apoptosis. CF also decreased the mitochondrial membrane potential in BGC-823 cells. Immunoblotting analysis revealed that CF significantly decreased the expressions of cyclinD1, Bcl-2, and Bcl-x(L), increased the expressions of cytosol cytochrome c, p53, p21, and Rb. In addition, CF significantly increased the expressions and activities of caspase-3 and -9. More importantly, CF potently inhibited the growth of BGC-823 cells xenografted in athymic nude mice with negligible body weight loss and damage towards the spleen. These results indicate that CF exerts a cytotoxic effect on BGC-823 cells by inducing cell cycle arrest and apoptosis.     

The apoptotic effect of shikonin on human papillary thyroid carcinoma cells through mitochondrial pathway

This study aims to explore the apoptotic function of shikonin on the papillary thyroid cancer cells and the related mechanism. The papillary thyroid cancer cell lines K1 and W3 and thyroid follicular epithelial cells NTHY-ORI 3-1 were treated with different concentrations of shikonin. Cell proliferation was tested. Morphological changes of the apoptotic cells were observed by Hoechst 33342 staining. The apoptosis rate of the papillary thyroid cancer cells was measured with flow cytometry. Changes of the cell cycle were explored. The mitochondrial membrane potential changes were analyzed after JC-1 staining. Bcl-2 family proteins and caspase-3 expression with shikonin treatment was analyzed by real-time fluorescence polymerase chain reaction (PCR). Cell proliferation of K1 and W3 was inhibited by shikonin, and the inhibition was dose–time dependent. Papillary thyroid carcinoma cells treated by shikonin had no obvious cell cycle arrest but were observed with the higher apoptosis rate and the typical apoptotic morphological changes of the cell nucleus. JC-1 staining showed that shikonin reduced the mitochondrial membrane potential of papillary thyroid carcinoma cells. Real-time PCR results showed that shikonin significantly increased Bax and caspase-3 expression and upregulated Bcl-2 expression in a dose-dependent manner in papillary thyroid carcinoma cells. However, the NTHY-ORI 3-1 was almost not affected by shikonin treatment. Shikonin can inhibit K1 and W3 cell proliferation in a dose- and time-dependent manner, enhance Bax levels, reduce anti-apoptotic protein Bcl-2 levels, result in decreasing mitochondrial membrane potential and activating caspase-3 enzyme, and finally lead to apoptosis.     

敲低EN2诱导肝癌细胞凋亡并提高PTEN蛋白的表达

 目的 探讨EN2对肝癌细胞凋亡及细胞中PTEN蛋白表达的影响。方法 用EN2 siRNA慢病毒感染肝癌细胞HuH-7，qRT-PCR和Western blot方法检测干扰效果。MTT方法测定细胞活力变化，PI单染法测定细胞周期分布，Annexin V-FITC/PI双染法测定细胞凋亡变化，Western blot测定细胞中激活型Caspase-3（Cleaved Caspase-3）、激活型Caspase-9（Cleaved Caspase-9）、第10号染色体同源缺失性磷酸酶-张力蛋白（PTEN）及细胞周期素B 1( Cyclin B l)蛋白水平，JC-1法测定线粒体膜电位变化，Western blot测定胞浆中细胞色素C（Cytochrome C）蛋白水平。结果 EN2 siRNA慢病毒感染可明显下调肝癌细胞中EN2的表达。沉默EN2表达后的肝癌细胞活力降低，细胞凋亡率升高，细胞G2/M期比例升高，细胞中Cleaved Caspase-3、Cleaved Caspase-9、PTEN蛋白水平明显升高，Cyclin B1蛋白水平明显降低，线粒体膜电位下降，胞质中Cytochrome C蛋白水平升高。结论 敲低EN2可以阻滞肝癌细胞周期，诱导肝癌细胞凋亡，其作用机制可能与PTEN、线粒体凋亡途径有关。     

Growth inhibition of colon cancer cells by polyisoprenylated benzophenones is associated with induction of the endoplasmic reticulum response

Polyisoprenylated benzophenones derived from Garcinia xanthochymus have cytotoxic activity in vitro and antitumor activity in rodent models, but the mechanism is unknown. The purpose of our study was to examine in parallel molecular pathways that are targeted by 3 Garcinia-derived benzophenones-xanthochymol (X), guttiferone E (GE) and guttiferone H (GH), in 3 human colon cancer cell lines, HCT116, HT29 and SW480. The IC50 concentrations were determined and the cells were then treated with X, GE or GH at their respective IC50 or IC50x2 concentrations. Effects on the cell cycle, mitochondrial membrane potential and apoptosis were assessed by flow cytometry and caspase activation. Changes in gene expression were assessed with Illumina 24 K gene arrays. We found that X, GE and GH induced loss of mitochondrial membrane potential and G1 arrest at their IC50 concentrations and induced caspase activation at IC50 x 2 concentrations. An analysis of the changes in gene expression revealed that with all 3 compounds and all 3 cell lines there was a marked increase in expression of several genes, including XBP1, ATF4 and DDIT3/CHOP, which are components of the endoplasmic reticulum stress response. The DDIT4/REDD1 gene, an inhibitor of the mTOR survival pathway, was also up-regulated. Therefore, X, GE and GH appear to inhibit the growth of human colon cancer cells, at least in part, by activating the endoplasmic reticulum stress response and inhibiting the mTOR cell survival pathway. These combined effects may contribute to the anticancer activity of these novel compounds.     

Magnolol, a natural compound, induces apoptosis of SGC-7901 human gastric adenocarcinoma cells via the mitochondrial and PI3K/Akt signaling pathways

Gastric cancer is the fourth most commonly diagnosed cancer with the second highest mortality rate worldwide. Surgery, chemotherapy and radiation therapy are generally used for the treatment of stomach cancer but only limited clinical response is shown by these therapies and still no effectual therapy for advanced gastric adenocarcinoma patients is available. Therefore, there is a need to identify other therapeutic agents against this life-threatening disease. Plants are considered as one of the most important sources for the development of anticancer drugs. Magnolol, a natural compound possesses anticancer properties. However, effects of Magnolol on human gastric cancer remain unexplored. The effects of Magnolol on the viability of SGC-7901 cells were determined by the MTT assay. Apoptosis, mitochondrial membrane potential and cell cycle were evaluated by flow cytometry. Protein expression of Bcl-2, Bax, caspase-3 and PI3K/Akt was analysed by Western blotting. Magnolol induced morphological changes in SGC-7901 cells and its cytotoxic effects were linked with DNA damage, apoptosis and S-phase arrest in a dose-dependent manner. Magnolol triggered the mitochondrial-mediated apoptosis pathway as shown by an increased ratio of Bax/Bcl-2, dissipation of mitochondrial membrane potential (ΔΨm), and sequential activation of caspase-3 and inhibition of PI3K/Akt. Additionally, Magnolol induced autophagy in SGC-7901 cells at high concentration but was not involved in cell death. Magnolol-induced apoptosis of SGC-7901 cells involves mitochondria and PI3K/Akt-dependent pathways. These findings provide evidence that Magnolol is a promising natural compound for the treatment of gastric cancer and may represent a candidate for in vivo studies of monotherapies or combination antitumor therapies.     

Calcium/calmodulin-dependent kinase inhibitor induces growth inhibition, cell cycle arrest, and apoptosis in human choriocarcinoma cells

KN-93, a membrane-permeant calcium/calmodulin- dependent kinase-selective inhibitor, induces apoptosis in some lines of human tumor cells. We investigated the effect of KN-93 in the choriocarcinoma cell line, BeWo. BeWo cells were treated with various concentrations of KN-93, and changes in cell growth, the cell cycle, apoptosis, and related parameters were examined. A WST-1 assay showed that BeWo cells were sensitive to the growth inhibitory effect of KN-93. Cell cycle analysis indicated that exposure to KN-93 decreased the proportion of cells in the S phase and increased the proportion in the G0/G1 phases of the cell cycle. Induction of apoptosis was confirmed by Annexin V staining of externalized phosphatidylserine, by the loss of mitochondrial transmembrane potential, and by antibodies directed against histones from fragmented DNA. This induction occurred in conjunction with the altered expression of genes related to cell growth, malignant phenotype, and apoptosis. These results suggest that KN-93 may serve as a therapeutic agent for the treatment of choriocarcinoma.     

Diosgenin induces cell cycle arrest and apoptosis in human leukemia K562 cells with the disruption of Ca2+ homeostasis

Purpose Diosgenin is a steroidal sapogenin with estrogenic and antitumor properties. In order to elucidate the mechanism of its antiproliferative activity, we investigated its effects on the cell cycle and apoptosis in human chronic myelogenous leukemia K562 cells.Methods Cell viability was assessed via an MTT assay. Apoptosis was investigated in terms of nuclear morphology, DNA fragmentation, and phosphatidylserine externalization. Cell cycle analysis was performed via PI staining and flow cytometry (FCM). Western blotting and immunofluorescence methods were used to determine the levels of p53, cell cycle-related proteins and Bcl-2 family members. FCM was also used to estimate the changes in mitochondrial membrane potential (MMP), intracellular Ca²⁺ concentration and reactive oxygen species (ROS) generation.Results Cell cycle analysis showed that diosgenin caused G₂/M arrest independently of p53. The levels of cyclin B1 and p21^Cip1/Waf1 were decreased, whereas cdc2 levels were increased. Subsequent apoptosis was demonstrated with the dramatic activation of caspase-3. A dramatic decline in intracellular Ca²⁺ concentration was observed as an initiating event in the process of cell cycle arrest and apoptosis, which was followed by the hyperpolarization and depolarization of MMP. Generation of ROS was observed in the progression of apoptosis. The antiapoptotic Bcl-2 and Bcl-x_L proteins were downregulated, whereas the proapoptotic Bax was upregulated.Conclusions Diosgenin inhibits K562 cell proliferation via cell cycle G₂/M arrest and apoptosis, with disruption of Ca²⁺ homeostasis and mitochondrial dysfunction playing vital roles.