Human hepatocellular carcinoma cells resist to TRAIL-induced apoptosis,and the resistance is abolished by cisplatin

TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, selectively induce apoptosis in various transformed cell lines but not in almost-normal tissues. It is regulated by 2 death receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2 and 2 decoy receptors, TRAIL-R3 and TRAIL-R4. However, the determining factors of the sensitivity to TRAIL-induced apoptosis are not clearly understood. Herein, we investigated the expression of TRAIL-R, c-FLIP, FADD-like interleukin-1beta-converting enzyme inhibitory protein, and TRAIL-induced apoptosis in human hepatocellular carcinoma (HCC) cell lines. Seven of ten HCC cell lines showed resistance to TRAIL-induced apoptosis and five of seven TRAIL-resistant cell lines became sensitive to TRAIL by co-treatment with cycloheximide. In HCC cell lines, their TRAIL resistance did not correlate with the basal expression level of TRAIL receptors or c-FLIP, however, in human tissues, TRAIL-R1 and TRAIL-R2 expressions were notably decreased compared to normal counterpart. Cisplatin showed synergistic effect on TRAIL-induced apoptosis in most HCC cell lines regardless of their p53 status and TRAIL-R1 was induced by cisplatin treatment in certain cell lines. Inhibition of nuclear factor K B (NF-kappaB) by SN50, a peptide inhibitor of NF-KB activity, had no effect on TRAIL-induced apoptosis in HCC cells. These results suggest that (a) the majority of human HCC cell lines are resistant to TRAIL-induced apoptosis and cycloheximide-sensitive short-lived antiapoptotic molecule(s) is responsible for this resistance, (b) the expression of TRAIL-R1 and TRAIL-R2 is reduced in HCC tissues, and the increased expression of TRAIL-R1 may be a mechanism of cisplatininduced sensitization to TRAIL-induced apoptosis in some HCC cells, and (c) the activation of NF-kappaB may not be involved in the TRAIL resistance of HCC cells     

Rapid detection of apoptosis in mammalian cells by using intact cell MALDI mass spectrometry

Detection of cell death has extensive applications and is of great commercial value. However, most current high-throughput cell viability assays cannot distinguish the two major forms of cell death: apoptosis and necrosis. Many apoptosis-specific detection methods exist but they are time consuming and labour intensive. In this work, we proposed a novel approach based on Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) for the specific detection of apoptosis in cultured mammalian cells. Buffer washed cells were directly mixed with a matrix solution and subsequently deposited onto the stainless steel target for MALDI analysis. The resulting mass spectrometric profiles were highly reproducible and can be used to reflect cell viability. Remarkably, the mass spectrometric profiles generated from apoptotic cells were distinct from those from either normal or necrotic cells. The apoptosis-specific features of the mass spectra were proportional to the percentage of apoptotic cells in the culture, but are independent of the drugs used to stimulate apoptosis. This is the first report on the utilization of intact cell MALDI mass spectrometry in detecting mammalian cell apoptosis, and can be used as a basis for the development of a reliable, fast, label-free and high-throughput method for detecting apoptotic cell death.     

Induction of Apoptosis by Ribosome Inactivating Proteins

Apoptotic cell death is a fundamental process in the development and physiological homeostasis of multicellular organisms. It is associated with control of cell numbers in tissues and organs during development, with cell turnover, and with response to infection. Molecules that trigger this process in continuously proliferating cancer cells can be used as chemotherapeutic agents. Ribosome inactivating proteins (RIPs) that inhibit translation in a cell by depurinating (N-glycosidase activity) the 28S rRNA are known to serve as apoptosis inducers. However, the role of depurination activity of the RIPs in apoptosis induction is still controversial. Presently, there are three different hypotheses which propose that depurination is: (1) essential, (2) essential but not the sole factor, or (3) not essential for apoptosis induction. This article reviews various experimental outcomes on the importance of N-glycosidase activity of RIPs in the induction of apoptosis.     

Isolation and Functional Characterization of Single Domain Antibody Modulators of Caspase-3 and Apoptosis

Apoptosis, or programmed cell death, is an essential process affecting homeostasis of cell growth, development, and the elimination of damaged or dangerous cells. Inappropriate cell death caused by oxidative stress has been implicated in the development of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and stroke. On the other hand, a defect in the cell death process leads to the development of cancer. For example, the main player of apoptosis, p53, is defective in many of the human cancers. Apoptosis is regulated by the interplay of pro-apoptotic and anti-apoptotic proteins from the Bcl-2 family and caspases. In particular, specific modulators of the activity of Caspase 3 could be very important for the development of therapies for diseases such as neurodegeneration and cancer. In this study, two V_HHs specific to Caspase 3 (VhhCasp31 and VhhCasp32) were isolated from a heavy chain antibody variable domain (V_HH) phage display library and tested for their apoptosis-modulating effects. While VhhCasp31 was found to be antagonistic towards Caspase 3, VhhCasp32 was agonistic. Furthermore, when expressed as intrabodies in SHSY-5Y neuroblastoma cells, VhhCasp31 rendered cells resistant to oxidative-stress-induced apoptosis, whereas VhhCasp32 resulted in apoptosis. These V_HH antagonist and agonist of apoptosis could have potential for the development of therapeutics for neurodegenerative diseases and cancer, respectively.     

Capillary electrophoretic determination of apoptosis of HeLa cells induced by trichosanthin

Apoptosis is a distinct mode of cell death that is responsible for deletion of cells in normal tissues; it also occurs in specific pathologic contexts. The observation of apoptosis is very important in the research of cancer and cancer therapy. The traditional observation method of apoptosis was agarose gel electrophoresis, which is depending on the determination of ladder-liking DNA fragments extracted from apoptotic cells. It is time-consuming and low-sensitive. Recently, the sieving capillary electrophoresis has been used to detect apoptosis too. However, the problem of DNA fragments contamination is still existing. Here, we have developed a capillary electrophoresis method that could detect apoptosis of whole cell directly and do not need to extract DNA fragments from cells. Apoptosis of adherent cell HeLa cell of carcinoma induced by cyclophosphamide was used as the model to establish the method. The effluence of medicine concentration on apoptosis of cells was studied in detail. It was also found that the method could detect the change of cells in the early period of apoptosis. The induction of apoptosis of HeLa cell by trichosanthin was determined with the method, and the result of flow cytometry was also proved that trichosanthin could result in apoptosis of HeLa cells.     

Paxilline enhances TRAIL-mediated apoptosis of glioma cells via modulation of c-FLIP, survivin and DR5

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells while sparing normal cells. However, many cancer cells are resistant to TRAIL-induced cell death. Here, we report that paxilline, an indole alkaloid from Penicillium paxilli, can sensitize various glioma cells to TRAIL-mediated apoptosis. While treatment with TRAIL alone caused partial processing of caspase-3 to its p20 intermediate in TRAIL-resistant glioma cell lines, co-treatment with TRAIL and subtoxic doses of paxilline caused complete processing of caspase-3 into its active subunits. Paxilline treatment markedly upregulated DR5, a receptor of TRAIL, through a CHOP/GADD153-mediated process. In addition, paxilline treatment markedly downregulated the protein levels of the short form of the cellular FLICE-inhibitory protein (c-FLIPs) and the caspase inhibitor, survivin, through proteasome-mediated degradation. Taken together, these results show that paxilline effectively sensitizes glioma cells to TRAIL-mediated apoptosis by modulating multiple components of the death receptor-mediated apoptotic pathway. Interestingly, paxilline/TRAIL co-treatment did not induce apoptosis in normal astrocytes, nor did it affect the protein levels of CHOP, DR5 or survivin in these cells. Thus, combined treatment regimens involving paxilline and TRAIL may offer an attractive strategy for safely treating resistant gliomas.     

The death of human cancer cells following photodynamic therapy: apoptosis competence is necessary for Bcl-2 protection but not for induction of autophagy

Photodynamic therapy (PDT) is an efficient inducer of apoptosis in many types of cells, except in cells deficient in one or more of the factors that mediate apoptosis. Recent reports have identified autophagy as a potential alternative cell death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145 cells) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v cells) and in apoptosis-competent cells (MCF-7c3 cells that stably overexpress human pro-caspase-3 and Chinese hamster ovary CHO 5A100 cells). Further, each of the cell lines was also studied with and without stably overexpressed Bcl-2. Autophagy was identified by electron microscopic observation of the presence of double-membrane-delineated autophagosomal vesicles in the cytosol and by immunoblot observation of the Pc 4-PDT dose- and time-dependent increase in the level of LC3-II, a component of the autophagosomal membrane. Autophagy was observed in all of the cell lines studied, whether or not they were capable of typical apoptosis and whether or not they overexpressed Bcl-2. The presence of stably overexpressed Bcl-2 in the cells protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO 5A100 cells). In contrast, Bcl-2 overexpression did not protect against the development of autophagy in any of the cell lines or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145 cells). Furthermore, 3-methyladenine and wortmannin, inhibitors of autophagy, provided greater protection against loss of viability to apoptosis-deficient than to apoptosis-competent cells. The results show that autophagy occurs during cell death following PDT in human cancer cells competent or not for normal apoptosis. Only the apoptosis-competent cells are protected by Bcl-2 against cell death.     

Molecular dynamics study of peptide segments of the BH3 domain of the proapoptotic proteins Bak, Bax, Bid and Hrk bound to the Bcl-xL and Bcl-2 proteins

Overexpression of Bcl-2 and Bcl-xL proteins, both inhibitors of apoptosis or programmed cell death, is related to the generation and development of several types of cancer as well as to an elevated resistance to chemotherapeutic treatments. Given that synthetic peptide fragments of the BH3 domain are capable to bind to both proteins and induce apoptosis in cell-free systems and HeLa cells, small molecule non-peptide mimics of these peptides can be considered as a new therapeutic strategy for the treatment of diseases associated to a deficient apoptosis or resistant to the treatments with chemotherapeutic drugs. This strategy is supported by experimental evidences about the death of transformed cells and sensibilization of tumoral cells by the inhibition of the antiapoptotic proteins Bcl-2 and Bcl-xL. In the current work, these proteins complexed with X(16BH3), where X designates the proapoptotic proteins Bak, Bax, Bid and Hrk, have been modeled in order to establish a pharmacophoric hypothesis that must be present in any ligand capable of binding with the antiapoptotic proteins Bcl-2 and Bcl-xL. The pharmacophore is also used to explain the structural features of a set of new small molecule inhibitors of these antiapoptotic proteins.     

Neuronal loss in primary long-term cortical culture involves neurodegeneration-like cell death via calpain and p35 processing, but not developmental apoptosis or aging

Primary neuronal culture is a powerful tool to study neuronal development, aging, and degeneration. However, cultured neurons show signs of cell death after 2 or 3 weeks. Although the mechanism underlying this phenomenon has not been elucidated, several preventive methods have been identified. Here we show that the neuronal loss in primary cortical culture involves calpain activation and subsequent neuronal cell death. Neuronal loss during cultivation showed destruction of neurites and synapses, and a decrease in neuron numbers. mu-Calpain and m-calpain were initially activated and accumulated by increased RNA expression. This neuronal death exhibited neurodegenerative features, such as conversion of p35 to p25, which is important in the developmental process and in the pathogenesis of Alzheimer's disease. But, postnatal and aged rat cortex did not show calpain activation and prolonged processing of p35 to p25, in contrast to the long-term culture of cortical neurons. In addition, the inhibition of calpains by ALLM or ALLN blocked the conversion of p35 to p25, indicating that the calpain activity is essential for the neurodegenerative features of cell death. Taken together, this study shows that the neuronal loss in primary cortical cultures involves neurodegeneration-like cell death through the activation of calpains and the subsequent processing of p35 to p25, but not developmental apoptosis or aging. Our results suggest that the long term primary culture of cortical neurons represent a valuable model of neurodegeneration, such as Alzheimer's disease.     

Cytotoxicity and Apoptosis Effects of Curcumin Analogue (2E,6E)-2,6-Bis(2,3-Dimethoxybenzylidine) Cyclohexanone (DMCH) on Human Colon Cancer Cells HT29 and SW620 In Vitro

Colorectal cancer (CRC) is the third most common type of cancer worldwide and a leading cause of cancer death. According to the Malaysian National Cancer Registry Report 2012–2016, colorectal cancer was the second most common cancer in Malaysia after breast cancer. Recent treatments for colon cancer cases have caused side effects and recurrence in patients. One of the alternative ways to fight cancer is by using natural products. Curcumin is a compound of the rhizomes of Curcuma longa that possesses a broad range of pharmacological activities. Curcumin has been studied for decades but due to its low bioavailability, its usage as a therapeutic agent has been compromised. This has led to the development of a chemically synthesized curcuminoid analogue, (2E,6E)-2,6-bis(2,3-dimethoxybenzylidine) cyclohexanone (DMCH), to overcome the drawbacks. This study aims to examine the potential of DMCH for cytotoxicity, apoptosis induction, and activation of apoptosis-related proteins on the colon cancer cell lines HT29 and SW620. The cytotoxic activity of DMCH was evaluated using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) cell viability assay on both of the cell lines, HT29 and SW620. To determine the mode of cell death, an acridine orange/propidium iodide (AO/PI) assay was conducted, followed by Annexin V/FITC, cell cycle analysis, and JC-1 assay using a flow cytometer. A proteome profiler angiogenesis assay was conducted to determine the protein expression. The inhibitory concentration (IC₅₀) of DMCH in SW620 and HT29 was 7.50 ± 1.19 and 9.80 ± 0.55 µg/mL, respectively. The treated cells displayed morphological features characteristic of apoptosis. The flow cytometry analysis confirmed that DMCH induced apoptosis as shown by an increase in the sub-G0/G1 population and an increase in the early apoptosis and late apoptosis populations compared with untreated cells. A higher number of apoptotic cells were observed on treated SW620 cells as compared to HT29 cells. Human apoptosis proteome profiler analysis revealed upregulation of Bax and Bad proteins and downregulation of Livin proteins in both the HT29 and SW620 cell lines. Collectively, DMCH induced cell death via apoptosis, and the effect was more pronounced on SW620 metastatic colon cancer cells, suggesting its potential effects as an antimetastatic agent targeting colon cancer cells.     

PHOTODYNAMIC THERAPY WITH PHOTOFRIN II INDUCES PROGRAMMED CELL DEATH IN CARCINOMA CELL LINES

Abstract The mode of cell death following photodynamic therapy was investigated from the perspective of programmed cell death or apoptosis. Human prostate carcinoma cells (PC3), human non-small cell lung carcinoma (H322a) and rat mammary carcinoma (MTF7) were treated by photodynamic therapy. An examination of extracted cellular DNA by gel electrophoresis showed the characteristic DNA ladder indicative of internucleosomal cleavage of DNA during apoptosis. The magnitude of the response and the photodynamic therapy dosage required to induce DNA fragmentation were different in PC3 and MTF7. The MTF7 cells responded with rapid apoptosis at the dose of light and drug that yielded 50% cell death (LD₅₀). In contrast, PC3 showed only marginal response at the LD₅₀ but had a marked response at the LD₈₅. Thus, apoptosis did not ensue as quickly in PC3 as in MTF7. The H322a cells were killed by photodynamic therapy but failed to exhibit any apoptotic response. The results also suggested that apoptosis in these cell lines has a minor requirement for de novo protein synthesis and no requirement for de novo RNA synthesis. This study indicates that although apoptosis can occur during photodynamic therapy-induced cell death, this response is not universal for all cancer cell lines.     

Synthesis,characterization and biological evaluation of some novel sulfonylthiosemicarbazides

Abstract

A series of thiosemicarbazides were synthesized and structurally characterized by spectroscopic techniques (NMR, FT-IR) besides elemental analysis. These compounds were evaluated for their cytotoxicity against human breast cancer cell line MCF7 and prostate cancer cell line PC3 and nonmalignant fibroblast L929 cell line by MTT assay. Among the compounds, N-[2-(4-chlorophenyl)ethyl]-2-[(4-methylphenyl)sulfonyl]hydrazinecarbothioamide (3d) and 2-[(4-methylphenyl)sulfonyl]-N-[4-(trifluoromethoxy)phenyl]hydrazinecarbothioamide (3f) were found to display significant cytotoxicity with IC₅₀ of 13.87?μM (against PC3 cell line) and 1.47?μM (against MCF7 cell line), respectively. These compounds were non-cytotoxic to normal cell line with IC₅₀>100?μM. Western blotting studies demonstrated that compound 3f induced apoptosis and caused cell death in the MCF7 and PC3 cell lines via an increase in Bax protein expression and a slight decrease in Bcl-2 protein expression. The gene expression ratio Bax/Bcl-2 showed the induction of mitochondrial apoptosis in cancer cell lines. All of synthesized compounds have also been tested for antioxidant activity and all compounds achieved strong inhibition of the DPPH radical. These findings showed that compound 3f, displays potential to be further explored in the development of new anticancer agents.     

Anti-neoplastic effects of gallic acid, a major component of Toona sinensis leaf extract, on oral squamous carcinoma cells

Extract of Toona sinensis (TS) has been reported to have various effects on cultured cell lines, including anti-proliferative activity in cancer cells. We have studied the effects of TS on various human oral squamous carcinoma cell lines (HOSCC), including UM1, UM2, SCC-4, and SCC-9. These cell lines were treated with TS leaf extract and screened for viability, apoptosis, necrosis, and apoptotic gene expression. Normal human oral keratinocytes (NHOK) served as a control for cytotoxic assays. Viability of TS-treated HOSCC was reduced, whereas that of NHOK was not affected. FACScan analysis revealed that the leaf extract induced apoptosis or a combination of apoptosis and necrosis, depending on cell type. Microarray and semi-quantitative RT-PCR analysis for apoptotic-related gene expression revealed that 3,4,5-trihydroxybenzoic acid (gallic acid, one of the major bioactive compounds purified from TS extract) up-regulated pro-apoptotic genes such TNF-α, TP53BP2, and GADD45A, and down-regulated the anti-apoptotic genes Survivin and cIAP1, resulting in cell death. This study suggests that gallic acid, the major bioactive compound present, is responsible for the anti-neoplastic effect of Toona sinensis leaf extract.     

Structural studies of apoptosis and ion transport regulatory proteins in membranes

Solid-state NMR spectroscopy is being used to determine the structures of membrane proteins involved in the regulation of apoptosis and ion transport. The Bcl-2 family includes pro- and anti-apoptotic proteins that play a major regulatory role in mitochondrion-dependent apoptosis or programmed cell death. The NMR data obtained for (15)N-labeled anti-apoptotic Bcl-xL in lipid bilayers are consistent with membrane association through insertion of the two central hydrophobic alpha-helices that are also required for channel formation and cytoprotective activity. The FXYD family proteins regulate ion flux across membranes, through interaction with the Na(+), K(+)-ATPase, in tissues that perform fluid and solute transport or that are electrically excitable. We have expressed and purified three FXYD family members, Mat8 (mammary tumor protein), CHIF (channel-inducing factor) and PLM (phospholemman), for structure determination by NMR in lipids. The solid-state NMR spectra of Bcl-2 and FXYD proteins, in uniaxially oriented lipid bilayers, give the first view of their membrane-associated architectures.     

1,4-Diselenophene-1,4-diketone triggers caspase-dependent apoptosis in human melanoma A375 cells through induction of mitochondrial dysfunction

Epidemiological, preclinical and clinical studies have supported the role of selenocompounds as potential cancer chemopreventive and chemotherapeutic agents. In this study, a novel selenophene-based compound, 1,4-diselenophene-1,4-diketone (DSeD), has been synthesized by Double Friedel-Crafts reaction and identified as a potent antiproliferative agent against a panel of six human caner cell lines. Despite this potency, DSeD was relatively nontoxic toward human normal cells, HS68 fibroblasts and HK-2 kidney cells. These results suggest that DSeD possesses great selectivity between cancer and normal cells. Induction of apoptosis in human melanoma A375 cells by DSeD was evidenced by accumulation of sub-G1 cell population, DNA fragmentation and nuclear condensation. Activation of caspase-9 and depletion of mitochondrial membrane potential indicated the initiation of the mitochondria-mediated apoptosis pathway. Pretreatment of cells with general caspase inhibitor z-VAD-fmk and caspase-9 inhibitor z-LEHD-fmk significantly suppressed the cell apoptosis, demonstrating the important roles of caspase and mitochondria in DSeD-induced apoptotic cell death. Furthermore, DSeD-induced apoptosis was found independent of reactive oxygen species generation. Taken together, our results suggest that DSeD induces caspase-dependent apoptosis in A375 cells through activation of mitochondria-mediated apoptosis pathway.     

Signaling pathways in cell death and survival after photodynamic therapy

Photodynamic therapy (PDT) is a cytotoxic treatment, which can induce cells to initiate a rescue response, or to undergo cell death, either apoptosis or necrosis. The many signaling pathways involved in these processes are the topic of this review. The subcellular localization of the photosensitizer has been shown to be a key factor in the outcome of PDT. Mitochondrial localized photosensitizers are able to induce apoptosis very rapidly. Lysosomal localized photosensitizers can elicit either a necrotic or an apoptotic response. In the plasma membrane, a target for various photosensitizers, rescue responses, apoptosis and necrosis is initiated. Several protein phosphorylation cascades are involved in the regulation of the response to PDT. Finally, a number of stress-induced proteins play a role in the rescue response after PDT. Notably, the induction of apoptosis by PDT might not be crucial for an optimal outcome. Recent studies indicate that abrogation of the apoptotic pathway does alter the clonogenic survival of the cells after PDT. Further studies, both in vitro and especially in vivo could lead to more efficient combination therapies in which signaling pathways, involved in cell death or rescue, are either up- or downregulated before PDT.     

Apoptosis,Paraptosis and Autophagy: Death and Survival Pathways Associated with Photodynamic Therapy

The ability of photosensitizing agents to create photodamage at specific subcellular sites has proved useful for characterizing pathway(s) to cell death and for selecting optimal targets for anti‐tumor efficacy. Both apoptosis and autophagy can occur after photodamage directed at mitochondria, lysosomes or the ER, with the balance often a determinant of overall efficacy. A combination of lysosomal + mitochondrial targets is associated with enhanced efficacy. More recently, ER photodamage was found to evoke a mainly unexplored mode of photokilling that involves extensive cytoplasmic vacuole formation but does not represent autophagy. This has been termed “paraptosis” and appears to be a reaction to the appearance of misfolded ER proteins. This report is designed to summarize current knowledge relating to death pathways and update information relating to paraptosis as a PDT response.     

Optogenetic Apoptosis: Light‐Triggered Cell Death

An optogenetic Bax has been designed that facilitates light‐induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light‐responsive Bax results in the release of mitochondrial proteins, downstream caspase‐3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C‐terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax‐mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis‐induced channel analogous to that of endogenous Bax.     

Apoptotic cell death dynamics of HL60 cells studied using a microfluidic cell trap device

This paper presents the design, fabrication and first results of a microfluidic cell trap device for analysis of apoptosis. The microfluidic silicon-glass chip enables the immobilization of cells and real-time monitoring of the apoptotic process. Induction of apoptosis, either electric field mediated or chemically induced with tumour necrosis factor (TNF-alpha), in combination with cycloheximide (CHX), was addressed. Exposure of cells to the appropriate fluorescent dyes, FLICA and PI, allows one to discriminate between viable, apoptotic and necrotic cells. The results showed that the onset of apoptosis and the transitions during the course of the cell death cascade were followed in chemically induced apoptotic HL60 cells. For the case of electric field mediated cell death, the distinction between apoptotic and necrotic stage was not clear. This paper presents the first results to analyse programmed cell death dynamics using this apoptosis chip and a first step towards an integrated apoptosis chip for high-throughput drug screening on a single cellular level.