Caspase-3 Activity Analysis Research Articles

Microtubule‐binding protein CLIP‐170 is a mediator of paclitaxel sensitivity

CLIP-170 is a microtubule-binding protein and participates in diverse microtubule-associated cellular activities by regulating microtubule dynamics. Here we provide evidence that CLIP-170 is a mediator of the sensitivity of the anti-microtubule drug paclitaxel in breast cancer. In vitro cell proliferation assays reveal that CLIP-170 expression in breast cancer cell lines correlates with their sensitivity to paclitaxel. In addition, CLIP-170 expression in clinical samples of breast cancer correlates with the pathological response of tumours to paclitaxel-containing chemotherapy. Mitotic index and caspase-3 activity analyses reveal that CLIP-170 increases the abilities of paclitaxel to block cell cycle progression at mitosis and to induce apoptosis in breast cancer cells. By microtubule sedimentation assay and binding affinity analysis, we further find that CLIP-170 promotes paclitaxel binding to microtubules. In vitro tubulin polymerization assay shows that CLIP-170 enhances the activity of paclitaxel to promote microtubule assembly. These results demonstrate that CLIP-170 mediates paclitaxel sensitivity in breast cancer via a microtubule-dependent mechanism.

RNAi Screening Identifies TAK1 as a Potential Target for the Enhanced Efficacy of Topoisomerase Inhibitors

In an effort to develop strategies that improve the efficacy of existing anticancer agents, we have conducted a siRNA-based RNAi screen to identify genes that, when targeted by siRNA, improve the activity of the topoisomerase I (Top1) poison camptothecin (CPT). Screening was conducted using a set of siRNAs corresponding to over 400 apoptosisrelated genes in MDA-MB-231 breast cancer cells. During the course of these studies, we identified the silencing of MAP3K7 as a significant enhancer of CPT activity. Follow-up analysis of caspase activity and caspase-dependent phosphorylation of histone H2AX demonstrated that the silencing of MAP3K7 enhanced CPT-associated apoptosis. Silencing MAP3K7 also sensitized cells to additional compounds, including CPT clinical analogs. This activity was not restricted to MDA-MB-231 cells, as the silencing of MAP3K7 also sensitized the breast cancer cell line MDA-MB-468 and HCT-116 colon cancer cells. However, MAP3K7 silencing did not affect compound activity in the comparatively normal mammary epithelial cell line MCF10A, as well as some additional tumorigenic lines. MAP3K7 encodes the TAK1 kinase, an enzyme that is central to the regulation of many processes associated with the growth of cancer cells (e.g. NF- κB, JNK, and p38 signaling). An analysis of TAK1 signaling pathway members revealed that the silencing of TAB2 also sensitizes MDA-MB-231 and HCT-116 cells towards CPT. These findings may offer avenues towards lowering the effective doses of Top1 inhibitors in cancer cells and, in doing so, broaden their application.

Scorpion venom (Odontobuthus doriae) induces apoptosis by depolarization of mitochondria and reduces S-phase population in human breast cancer cells (MCF-7)

Venom of some species of scorpions induces apoptosis and arrests proliferation in cancer cells. This is an important property that can be harnessed and can lead to isolation of compounds of therapeutic importance in cancer research. Cytotoxicity was investigated using MTT reduction and confirmed with lactate dehydrogenase release following venom exposure. Apoptosis was evaluated with determination of mitochondrial membrane potential, reactive nitrogen species assay, measurement of Caspase-3 activity and DNA fragmentation analysis. To confirm that venom can inhibit DNA synthesis in proliferating breast cancer cells, immunocytochemical detection of BrdU incorporation was done. Our results demonstrated that venom of Odontobuthus doriae not only induced apoptosis but lead to the inhibition of DNA synthesis in human breast cancer cells (MCF-7). Cell viability decreased with parallel increment of LDH release in dose dependent manner after treatment with varying concentrations of venom. Moreover, venom depleted cellular antioxidants evidenced by depression of GSH and Catalases and concomitantly increased reactive nitrogen intermediates (RNI). These events were related to the depolarization of mitochondria and associated Caspase-3 activation following venom treatment in a concentration dependent manner. Finally, fragmentation of nuclear DNA following venom treatment confirmed the apoptotic property of the said venom. These results suggest that venom of O. doriae can be potential source for the isolation of effective anti-proliferative and apoptotic molecules.

The APP intracellular domain (AICD) potentiates ER stress-induced apoptosis

Here we employed human SHEP neuroblastoma cells either stably or inducibly expressing the amyloid precursor protein (APP) intracellular domain (AICD) to investigate its ability to modulate stress-induced cell death. Analysis of effector caspase activation revealed that AICD overexpression was specifically associated with an increased sensitivity to apoptosis induced by the 2 endoplasmic reticulum (ER) stressors thapsigargin and tunicamycin, but not by staurosporine (STS). Basal and ER stress-induced expression of Bip/Grp78 and C/EBP-homologous protein/GADD153 were not altered by AICD implying that AICD potentiated cell death downstream or independent of the conserved unfolded protein response (UPR). Interestingly, quantitative polymerase chain reaction analysis and reporter gene assays revealed that AICD significantly downregulated messenger RNA levels of the Alzheimer's disease susceptibility gene ApoJ/clusterin, indicating transcriptional repression. Knockdown of ApoJ/clusterin mimicked the effect of AICD on ER stress-induced apoptosis, but had no discernible effect on staurosporine-induced cell death. Our data suggest that altered levels of AICD may abolish the prosurvival function of ApoJ/clusterin and increase the susceptibility of neurons to ER stress-mediated cell death, a pathway that may contribute to the pathogenesis of Alzheimer's disease.

Real-time detection of cellular apoptosis using a rat C6 glioma cell-based assay system

Caspase-3 is a key mediator of apoptosis in mammalian cells. Cells expressing caspase-3 substrate peptides have become powerful and increasingly common components of cell-based assay systems. We developed a cell-based assay to measure staurosporine (STP)-induced caspase-3 activity in live rat glioma C6 cells. The caspase-3 sensing system was constructed to include a nuclear export signal (NES), followed by the amino acid sequence Asp-Glu-Val-Asp (DEVD) and a green fluorescent protein (GFP) fused to the Nterminal site of a nuclear localization signal (NLS). Using time-lapse confocal microscopy, we monitored caspase-3 activation during apoptosis by imaging the translocation of GFP from the cytosol to the nucleus. After 8 h of 0.5 μM STP treatment, caspase-3 activity was assessed by monitoring the translocation of GFP to the nucleus due to cleavage of the NES from the GFP by caspase-3. Finally, disintegration of the plasma membrane during late apoptosis was confirmed using a nuclear dye, propidium iodide. Analysis of caspase-3 activity using real-time monitoring can potentially be used to screen for apoptotic molecules in living cells.

Multimodal In Vivo Imaging and Blood Monitoring of Intrinsic and Extrinsic Apoptosis

Noninvasive detection and in vivo imaging of apoptosis plays a critical role in the development of therapeutics in many different fields including cancer. We have developed an apoptosis biosensor by fusing green fluorescent protein (GFP) to the N-terminus of the naturally secreted Gaussia luciferase separated by a caspase-3 cleavage peptide consisting of aspartic acid (D), glutamic acid (E), valine (V), and aspartic acid (D) or DEVD. We showed that this fusion is retained in the cytoplasm of cells in an inactive form. Upon apoptosis, the DEVD peptide is cleaved in response to caspase-3 activation, freeing ssGluc, which can now enter the secretory pathway where it is folded properly and is released from the cells and can be detected in the conditioned medium in culture or in blood of live animals ex vivo over time. Because Gluc is secreted from cells via conventional pathway through the endoplasmic reticulum (ER), Golgi and vesicles, we showed that the presence of Gluc in these compartments in response to apoptosis can be visualized in vivo using bioluminescence imaging. This reporter provides a valuable tool for imaging and real-time monitoring of apoptosis and is compatible with high-throughput functional screening application in cultured cells and animal models.

High Throughput Ratio Imaging to Profile Caspase Activity: Potential Application in Multiparameter High Content Apoptosis Analysis and Drug Screening

Recent advancement in the area of green fluorescent protein techniques coupled with microscopic imaging has significantly contributed in defining and dissecting subcellular changes of apoptosis with high spatio-temporal resolution. Although single cell based studies using EGFP and associated techniques have provided valuable information of initiation and hierarchical changes of apoptosis, they are yet to be exploited for multiparameter cell based real time analysis for possible drug screening or pathway defining in a high throughput manner. Here we have developed multiple cancer cell lines expressing FRET sensors for active caspases and adapted them for high throughput live cell ratio imaging, enabling high content image based multiparameter analysis. Sensitivity of the system to detect live cell caspase activation was substantiated by confocal acceptor bleaching as well as wide field FRET imaging. Multiple caspase-specific activities of DEVDase, IETDase and LEHDase were analysed simultaneously with other decisive events of cell death. Through simultaneous analysis of caspase activation by FRET ratio change coupled with detection of mitochondrial membrane potential loss or superoxide generation, we identified several antitumor agents that induced caspase activation with or without membrane potential loss or superoxide generation. Also, cells that escaped the initial drug-induced caspase activation could be easily followed up for defining long term fate. Employing such a revisit imaging strategy of the same area, we have tracked the caspase surviving fractions with multiple drugs and its subsequent response to retreatment, revealing drug-dependent diverging fate of surviving cells. This thereby indicates towards a complex control of drug induced tumor resistance. The technique described here has wider application in both screening of compound libraries as well as in defining apoptotic pathways by linking multiple signaling to identify non-classical apoptosis inducing agents, the greatest advantage being that the high content information obtained are from individual cells rather than being population based.

Transient Receptor Potential Melastatin 7 Channels are Involved in Ginsenoside Rg3-Induced Apoptosis in Gastric Cancer Cells

Ginsenosides play a role in a number of physiological and pharmacological functions in the gastrointestinal tract. The aim of this study was to clarify the potential role for transient receptor potential melastatin 7 (TRPM7) channels in ginsenoside Rg3-inhibited growth and survival of AGS cells, the most common human gastric adenocarcinoma cell line. The AGS cells were treated with varying concentrations of Rg3. Sub-G1 analysis, caspase-3 activity and poly(ADP-ribose) polymerase (PARP) cleavage analysis were conducted to determine whether AGS cell death occurs by apoptosis. TRPM7 channel blockers (La(3+) or 2-APB) and small interfering RNA (siRNA) were used in this study to confirm the role of TRPM7 channels. Furthermore, TRPM7 channels were over-expressed in human embryonic kidney (HEK) 293 cells to identify the role of TRPM7 channels in AGS cell growth and survival. The addition of Rg3 to the culture medium inhibited AGS growth and survival. Experimental results showed sub-G1 was markedly increased, caspase-3 activity was elevated, and degree of PARP cleavage was increased. TRPM7 channel blockade, either by La(3+) or 2-APB or by suppressing TRPM7 expression with siRNA, blocked the Rg3-induced inhibition of cell growth and survival. Furthermore, TRPM7 channel over-expression in HEK 293 cells exacerbated Rg3-induced cell death. These findings indicate that ginsenoside Rg3 inhibits the growth and survival of gastric cancer cell which is because of the blockade of TRPM7 channel activity. Therefore, TRPM7 channels may play an important role in the survival of gastric cancer.

Abstract 3262: Pharmaco-modulation of dihydroxy coumarins towards more selective antileukemic agents

Abstract Background: We described that 7,8-dihydroxy-4-methylcoumarin (DHMC) induces apoptosis in human leukemic U-937 cells, mediated by its pro-oxidant activity and activation of JNK. We also reported that DHMC shows selective toxicity for leukemic cells over normal peripheral blood monocytes. The aim of the present work is to establish the structural requirements for DHMC to display pro-apoptotic activity performing a structure activity relationship study of DHMC derivatives, cinnamic acid derivatives (as open-chain coumarin analogues) and a-pyrones (as representative of the d-lactone ring of the coumarin nucleus). Further, in an attempt to explain its selective toxicity, the mechanism of DHMC action through the JNK pathway will be characterized. Materials and Methods: DHMC analogues, cinnamic acid derivatives and a-pyrones were tested for their ability to induce biological effects in U-937 cells. Growth inhibition was determined through 3H-thymidine incorporation. Cellular viability was tested by trypan blue exclusion assays. Pro-apoptotic activity was evaluated by flow cytometry (annexin V), determination of caspase-3 activity and Western Blot analysis of caspase-3 and cleaved PARP levels. The oxidation potential and the capacity to generate free radicals were evaluated by cyclic voltammetry and electron paramagnetic resonance, respectively. To evaluate the role of cytoplasmic p21 (Cip1/WAF1) in the mechanism of DHMC selective action, we used U-937 cells transfected with a zinc-inducible p21 deletion mutant lacking the nuclear localization signal. Results: Only DHMC derivatives bearing two adjacent aromatic hydroxyl groups (catechol moiety) showed proliferation inhibition and pro-apoptotic activity in U-937 cells. a-pyrones had no effect on cellular viability, and cinnamic acid derivatives bearing the catechol moiety exhibited proliferation inhibition but no cytotoxic activity. Although ortho-dihydroxy coumarins and ortho-dihydroxy cinnamic acid derivatives showed a similar oxidation potential, only the former could generate stable free radicals in medium-like conditions, suggesting this capacity would be critical for their pro-apoptotic activity in U-937 cells. Finally, the pro-apoptotic effect of DHMC via JNK activation was abolished in U-937 cells expressing cytoplasmic p21, indicating this protein would be involved in the resistance that normal lymphocytes and monocytes, which normally express p21 in their cytoplasm, show for DHMC. Conclusions: DHMC-derived compounds carrying a catechol moiety can only induce apoptosis in U-937 cells if the integrity of the coumarinic nucleus is preserved. Selective toxicity of DHMC for leukemic cells would involve activation of JNK in the absence of cytoplasmic p21 expression. Collectively, our results point to ortho-dihydroxy coumarins as promising prototypes for the design of more selective antileukemic drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3262. doi:10.1158/1538-7445.AM2011-3262

Abstract 584: The novel proteasome inhibitors carfilzomib and ONX 0912 potently induce apoptosis and autophagy in head and neck squamous cell carcinoma cells via alteration of Bcl-2 family members

Abstract The goal of this study was to investigate the impact and molecular mechanism of action of two novel proteasome inhibitors, carfilzomib (also named PR-171) and ONX 0912 (previously named PR-047), in head and neck squamous cell carcinoma (HNSCC) cells. HNSCC is the sixth most common cancer in the United States, and is frequently characterized by resistance to conventional chemotherapy. Previously we have shown that HNSCC cells are highly sensitive to the proteasome inhibitor bortezomib. However, clinical application of bortezomib against solid tumors has been hampered by issues of nonspecificity and adverse cytotoxicities. In the current study we examined the effects of two next generation proteasome inhibitors with improved specificities for the chymotrypsin-like activity of the proteasome. Carfilzomib is a well-tolerated epoxyketone, and ONX 0912 is an orally bioavailable derivative. Carfilzomib and ONX 0912 exhibited potent killing activity against eight different HNSCC cell lines. In 48-hour trypan blue exclusion assays IC50 values for carfilzomib ranged from 18.3-70.4 nM, while IC50 values for ONX 0912 ranged from 58.9-185.7 nM. Carfilzomib and ONX 0912 activated HNSCC apoptosis signaling as demonstrated by flow cytometric analysis of Annexin V/PI staining and immunoblot analysis of caspase-3 activation and PARP cleavage. Treatment with carfilzomib or ONX 0912 also led to substantial alterations in the expression levels of Bcl-2 family members, including downregulation of anti-apoptotic Bcl-2 and upregulation of pro-apoptotic Bik, Bim, and Bax; these effects likely serve to promote cell killing by these compounds. At the same time, treatment also led to dramatic upregulation of anti-apoptotic Mcl-1L, and moderate upregulation of anti-apoptotic Bcl-XL; these effects may act to blunt the killing activities of the compounds. Additional studies revealed that carfilzomib and ONX 0912 promote autophagy in HNSCC cells. In HNSCC cells engineered to express GFP-LC3, treatment led to discrete cytoplasmic puncta characteristic of autophagasome formation. Moreover, carfilzomib and ONX 0912 promoted upregulation of Beclin-1, ATG-5, and LC3-II, indicative of autophagy induction. The induction of autophagy by carfilzomib and ONX 0912 was associated with upregulation of JNK enzymes and phosphorylation of Bcl-2. Taken together, our findings demonstrate that carfilzomib and ONX 0912 activate both apoptosis and autophagy signaling in HNSCC cells. In view of the potent killing activities of these compounds against HNSCC cells, carfilzomib and ONX 0912 may represent viable therapeutic agents in this malignancy. Further studies using HNSCC in vivo models are clearly warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 584. doi:10.1158/1538-7445.AM2011-584

Effects of dehydroepiandrosterone on proliferation, migration, and death of breast cancer cells

Cancer invasion and metastasis are the leading causes of mortality in patients with breast cancer. Dehydroepiandrosterone (DHEA) has a protective role against cancer, however, the mechanism by which DHEA has this effect remains poorly understood. The present study was aimed at investigating the actions of DHEA on the proliferation, cell cycle, death and migration of breast cancer cell lines. We used MCF-7 cells (estrogen receptors positive) and MDA-MB-231 and Hs578T cells (estrogen receptors negative) for these studies. Cell proliferation was evaluated by crystal violet staining, cell cycle by flow cytometry, and cell death by the carboxyfluorescein FLICA analysis of caspase activation. Migration was evaluated by transwell cell migration and wound assay. We also determined the expression of ECM-1 protein by western blotting and RT-PCR in real time. DHEA inhibited the proliferation of all breast cancer cell lines. This was associated with an arrest in the G1 phase of the cell cycle and death in MCF-7 cells. There was no alteration in any of the cell cycle phases or death in DHEA treated MDA-MB-231 or Hs578T cells. DHEA also suppressed the migration of all breast cancer cell lines, independently of the presence of estrogen receptors and decreased the expression of ECM-1 protein in Hs578T cells. These results suggest that the mechanism of DHEA actions against breast cancer involves the inhibition of cell proliferation and the suppression of migration, indicating that DHEA could be useful in the treatment of breast cancer.

Gallic Acid Isolated from Pomegranate Peel Extract Induces Reactive Oxygen Species Mediated Apoptosis in A549 Cell Line

Antioxidant properties elicited by plant species have a full range of perspective applications in human health care. In recent years, the prevention of cancer and cardiovascular diseases has been associated with the ingestion of fresh fruits, vegetables or teas rich in natural antioxidants [1]. Extracts from the different part of the pomegranate plant such as juice, seed and peel have been reported to exhibit a potent antioxidant activity. But the anticarcinogenic activity of active principle from the pomegranate peel extract was not studied so far. Hence the present study was planned to explore the molecular mechanism of the anticarcinogenic activity pomegranate peel on A549 cell line. In this study, GC-MS analysis was carried out for the methanolic extract of pomegranate peel which revealed gallic acid (GA) as the major antioxidant compound in the extract. Hence GA was purified further through RP-HPLC and evaluated its anticancer potential by studying its effect on mitochondrial respiration, cell-membrane integrity, apoptotic body formation and the DNA fragmentation in cultured A549 cells. We observed increased level of reactive oxygen species in the cells treated with GA at the concentrations of 10 and 20 ug/ml. Further analysis of caspase activation (caspase 8 and 9) revealed activation of caspases 9 in the cells treated with GA at a concentration of 20 ug/ml. Thus the present study revealed that the GA isolated from the pomegranate peel extract (Kabul variety) induced apoptosis in A549 cells through intrinsic pathway.

Effect of Curcumin on Doxorubicin-induced Cytotoxicity in H9c2 Cardiomyoblast Cells

Objective(s) Doxorubicin (DOX), a widely used chemotherapeutic agent can give rise to serve cardiotoxicity by inducing apoptosis. Curcumin, the active compound of the rhizome of Curcuma longa L. has anti-inflammatory, antioxidant and anti-proliferative activities. Curcumin has been identified to increase cytotoxicity in several cancer cell lines in combination with DOX, but there is no study about its effect and DOX on normal cardiac cells. Therefore, in the present study, we evaluated the effect of curcumin on apoptosis induced by DOX in H9c2 rat heart-derived cells. Materials and Methods Cell viability was determined by MTT assay. Also, activation of caspase-3 was evaluated by spectrophotometry. Quantitative real time RT-PCR was used to evaluate the expression of c-IAP1. Detection of intracellular DOX accumulation was performed by flow cytometry. Results No toxicity observed when the cells exposed for 1 hr to different concentrations of curcumin, but pretreatment of cells with curcumin increased cytotoxicity of DOX in a dose dependent manner. Analysis of caspase-3 activation showed that curcumin pretreatment increased caspase-3 activation. RT-PCR analysis clearly showed that curcumin significantly decreased mRNA gene expression of c-IAP1 compared to cells treated with DOX alone. Pretreatment of H9c2 cells with DOX and curcumin had no effect on the intracellular accumulation of DOX. Conclusion Our observations indicated that subtoxic concentrations of curcumin sensitize H9c2 cells to DOX-induce apoptosis. These results suggest that the use of curcumin in combination with DOX in malignancy must be reevaluated.

Abstract B52: Evaluation of role of apoptosis and cell cycle analysis in chemopreventive action of fish oil and celecoxib in the initiation phase of experimental mammary carcinogenesis

Abstract Aim: Cyclooxygenase (COX)-2 enzymes plays an important role in the progression and metastasis of cancer. COX −2 inhibition by non steroidal anti inflammatory drugs (NSAID) is a useful approach for cancer prevention but has side effects. On the other hand, n-3 polyunsaturated fatty acids (PUFA) also have a cancer chemopreventive effect mediated by COX-2 inhibition. Therefore, the present study was designed to investigate the chemopreventive effect of combined dosage of n-3 PUFA rich fish oil and celecoxib, a COX-2 inhibitor in the initiation phase of experimental mammary carcinogenesis. Study Design: Female Wistar rats were distributed into six groups: group-1 (vehicle treated), group-2 (DMBA treated), group-3 (20mg/kg body weight celecoxib), group-4 (0.5 ml fish oil), group-5 (20 mg/kg body weight celecoxib+0.5 ml fish oil) and group-6 (20 mg/kg body weight celecoxib+0.5 ml fish oil). The treatment was given for seven days and on 8th day animals in all groups except group 1 and 5 received an oral dose of 15mg of DMBA (7, 12-Dimethylbenz (a) anthracene) and sacrificed after 90 days. Histopathology was done for morphological examination of the tissue specimens. Apoptosis was measured by using M30 cytodeath monoclonal antibody and Caspase-3 activity and cell cycle analysis by propidium iodide (PI) staining was performed in isolated mammary epithelial cells. Results: In the animals treated with DMBA, the mammary tissue showed the features of ductal hyperplasia. Treatment with combination dosages of celecoxib+fish oil+DMBA resulted in a histological profile similar to the normal mammary tissue while obstruction in duct and lobule as well as hyperplastic changes were observed in the mammary tissue of celecoxib or fish oil treated animals. Group 2 animals showed a significant increase in apoptosis with M-30 cytoDEATH and caspase-3 activity. In addition, an increase in cell population in S and G2/M phase with a concomitant decrease in G1 phase cells was observed in carcinogen treated animals. In comparison to DMBA treated animals, a decrease in percentage apoptotic cells was observed in celecoxib or/and fish oil pretreated groups followed by DMBA with the least reduction in combinatorial regimen treated animals. There was no significant difference in the Caspase-3 activity in the DMBA treated animals and celecoxib + fish oil + DMBA treated animals. In the animals treated with combination dosage of celecoxib + fish oil + DMBA, there was a significant increase in apoptosis as compared to animals treated with celecoxib and fish oil alone. The apoptosis in the groups was also significantly increased with respect to control animals (group 1). Conclusion: Though both celecoxib and fish oil showed chemopreventive potential in experimental mammary carcinogenesis, better efficacy was observed with the combinatorial treatment and their synergistic effect may be mediated by the apoptotic pathway. Citation Information: Cancer Prev Res 2010;3(12 Suppl):B52.

Intact Apoptosis Signaling In Pediatric ALL Xenograft Leukemia Is Associated with Favorable Patient Outcome, Prolonged NOD/SCID Engraftment and Low Expression of Anti-Apoptotic Molecules

AbstractAbstract 2722Defects in cell death signaling might be responsible for treatment failure and development of relapse in acute leukemia. In two retrospective studies we recently investigated the functional integrity of apoptosis signaling in primary patient leukemia samples and reported an importance of intact apoptosis signaling for good outcome in pediatric ALL and AML. Using a NOD/SCID/huALL mouse xenotransplant model for pediatric ALL we have also shown that rapid engraftment of primary ALL cells (short time to leukemia/TTLshort) is characteristic for early relapse of the corresponding patient.The aim of this prospective study was to analyze the impact of the functional integrity of apoptosis signaling in xenograft ALL samples on NOD/SCID engraftment and patient outcome. Furthermore, expression of apoptosis regulating molecules was investigated and correlated to NOD/SCID engraftment, response to treatment and apoptosis signaling.Primary BCP-ALL samples (N=20) obtained at diagnosis were transplanted onto NOD/SCID mice and time to leukemia (TTL) was estimated as weeks from transplant to onset of disease for each sample transplanted. Apoptosis signaling was investigated in xenograft leukemia samples analyzing two key apoptogenic events by flowcytometry, cytochrome c release and caspase-3 activation. Transcript levels of the anti-apoptotic molecules Mcl-1, XIAP, Bcl-2 and Livin was analyzed by quantitative RT-PCR.Of the 20 ALL samples transplanted 6 led to rapid leukemia manifestation in the recipient animals (TTLshort) with an inferior relapse free survival of the corresponding patients in contrast to 14 patients with TTLlong phenotype (log rank P.002).Apoptosis signaling was investigated in xenograft leukemia samples by analysis of caspase-3 activation and cytochrome c release. Both events were closely correlated to each other indicating intact signaling in xenograft leukemia derived from patients stratified into non-high risk groups, patients showing good response to treatment (remission on day 15, negative MRD on day 33), and patients without relapse. In contrast, no correlation was found in xenografts from patients with poor outcome. Most importantly, intact apoptosis signaling was also found in TTLlong but not in TTLshort patients strongly indicating that mitochondrial cytochrome c release and consecutive apoptosome formation resulting in activation of downstream effector caspases such as caspase-3 is characteristic for a long engraftment phenotype and prognostic favorable ALL.The functional integrity of this apoptogenic checkpoint is subsumed by the parameter cytochrome c related activation of caspases (CRAC). Patients with positive CRAC values reflecting intact apoptosis signaling showed a significantly superior relapse free survival in contrast to patients with disturbed cytochrome c related caspase activation/negative CRAC (log rank, P<.001).Transcript expression of apoptosis regulating molecules was analyzed. A correlation of cytochrome c release and caspase-3 activation indicating proficient apoptosis signaling was exclusively observed in leukemia samples with low expression of the anti-apoptotic molecules Mcl-1, XIAP, Bcl-2 and Livin. Furthermore, patients samples with low Mcl-1 expression showed a significantly longer time to leukemia (TTL) and lower blast cells on day 8 than samples with high Mcl-1 expression.In summary, the propensity of leukemia cells to undergo apoptosis (CRAC positive) leads to prolonged engraftment upon transplant in the NOD/SCID/huALL model (TTLlong), is associated with low expression of anti-apoptotic molecules, and results in favorable treatment response and superior survival of pediatric ALL patients. Disclosures:No relevant conflicts of interest to declare.

Antiproliferative and pro-apoptotic effects afforded by novel Src-kinase inhibitors in human neuroblastoma cells

BackgroundNeuroblastoma (NB) is the second most common solid malignancy of childhood that usually undergoes rapid progression with a poor prognosis upon metastasis. The Src-family tyrosine kinases (SFKs) are a group of proteins involved in cancer development and invasiveness that seem to play an important role in the NB carcinogenesis.MethodsTo determine cell proliferation, the growth rate was evaluated by both MTT test and cells counted. Analysis of DNA content was performed for the evaluation of the cell cycle and apoptosis. To characterize the mechanisms underlying the antiproliferative effects induced by SI 34, a novel pyrazolo-pyrimidine derivative provided with Src inhibitory activity, the involvement of some cellular pathways that are important for cell proliferation and survival was investigated by western blot assays. In particular, the contribution of cyclins, Src and ERK were examined. Finally, experiments of cell adhesion and invasiveness were performed.ResultsTreatment of SH-SY5Y human NB cells and CHP100 human neuroepithelioma (NE) cultures with three novel pyrazolo[3,4-d]pyrimidine derivatives, namely SI 34, SI 35 and SI 83, inhibits the cell proliferation in a time and concentration-dependent manner. The maximal effect was obtained after 72 hours incubation with SI 34 10 μM. Fluorescence microscopy experiments, flow cytometry analysis and determination of caspase-3 activity by fluorimetric assays showed that SI 34 induced SH-SY5Y apoptosis. Moreover, SI 34 determined cell cycle arrest at the G0/G1 phase, paralleled by a decreased expression of cyclin D1. Furthermore, our data indicate that SI 34 reduces the SH-SY5Y cells adhesion and invasiveness. Evidence that SI 34 inhibits the Src and the ERK-phosphorylation, suggests the mechanism through which it exerts its effects in SH-SY5Y cells.ConclusionsOur study shows the ability of this pyrazolo-pyrimidine Src inhibitor in reducing the growth and the invasiveness of human NB cells, suggesting a promising role as novel drug in the treatment of neuroblastoma.

Organogallium(III) complexes as apoptosis promoting anticancer agents for head and neck squamous cell carcinoma (HNSCC) cell lines

Organogallium(III) dinuclear (1–9) and tetranuclear (10) complexes present potential therapeutic agents for the treatment of various types of cancer. The antiproliferative activity of 1–10 was evaluated with cell lines of head and neck squamous cell carcinomas, e.g. HN (soft palate), Cal27, Cal33 (tongue) and FaDu (hypopharynx) cell lines. The activity of compound 8 is comparable with that of cisplatin on cell line Cal27 (IC50 4.6μM for both compounds). The mode of cell death induced, caspase activity and cell cycle analysis were evaluated for potential hit compounds 3, 5 and 8 Potential hit compounds 3, 5 and 8 were further evaluated for the mode of cell death, caspase activity and cell cycle analysis. Apoptosis induced by compounds 3, 5 and 8 on Cal27 and FaDu cells was confirmed by DNA laddering , as well as acridine orange (AO) and ethidium bromide (EB) double staining. These compounds (3, 5 and 8) induced caspase-independent apoptosis (within 4h of action) in cell line Cal27. Extrinsic-mediated apoptosis associated with caspase 8 and 3 activation is the main mode of cytotoxicity induced on FaDu cells by compounds 3, 5 and 8. Cell cycle perturbations caused by these compounds are also observed. Our data suggest that compounds 3, 5 and 8 should be studied further for the treatment of head and neck cancer.

P06. Live-imaging analysis of apoptosis and caspase activation reveals that apoptosis is a facilitator of the neural tube closure

Neural tube closure (NTC) is a fundamental process to construct the brain and the spinal cord in vertebrates. The neural plate develops bilateral neural folds, which in turn come into contact in the midline and fuse together to create the neural tube. Programmed cell death (PCD), especially apoptosis, is widely observed during the process. Several lines of evidence suggested that apoptosis is essential to complete cranial NTC. However, it is still unclear how apoptosis contributes to the completion of cranial NTC. To examine the relationship between dying cells and morphogenetic movement such as neural plate bending, fusion of neural ridge, and epithelial remodeling after the fusion, direct visualization of dying cells during NTC could be effective. We have conducted a live-imaging analysis of apoptosis during cranial NTC with the mouse embryo expressing SCAT3, a fluorescent indicator protein to monitor caspase-3 activation in living cells. In these embryos, caspase activation was observed in dying cells of the pre-fusion neural ridge, fusion point, and the post-fusion midline. Thus, this system allowed us to observe the dynamics of cranial NTC when caspase activation and apoptosis were inhibited. We found that the speed of closure tended to become slow under caspase-inhibited condition, suggesting that caspase activation and apoptosis contribute to the progression of neural tube closure within a limited developmental time window.

SLC4A7 sodium bicarbonate co-transporter controls mitochondrial apoptosis in ischaemic coronary endothelial cells

Bicarbonate transport has been shown to participate in apoptosis under ischaemic stress. However, the precise transporting mechanisms involved in ischaemic apoptosis are unknown and were thus the aim of the present study. Rat coronary endothelial cells (EC) were exposed to simulated in vitro ischaemia for 2 h, and apoptosis was subsequently determined by chromatin staining and caspase-3 activity analysis. By examining the expression of bicarbonate transporters (BT) in EC by reverse transcriptase polymerase chain reaction and western blotting, a marked expression of the electroneutral sodium bicarbonate co-transporter (SLC4A7) was defined. To analyse the potential role of this transporter during apoptosis, a selective inhibitor (S0859, Sanofi-Aventis) was applied. Treatment with S0859 significantly increased caspase-3 activity and elevated the number of apoptotic EC. These results were comparable with an unselective inhibition of all BT due to withdrawal of bicarbonate in the anoxic medium. Knockdown of SLC4A7 in EC by transfecting appropriate siRNA similarly increased apoptosis of EC under simulated ischaemia. The initial characterization of the participating mechanisms of SLC4A7-dependent apoptosis revealed an activation of the mitochondrial pathway of apoptosis, i.e. cleavage of caspase-9 and binding of Bax to mitochondria. In contrast, no activation of the endoplasmic reticulum-dependent pathway (caspase-12 cleavage) or the extrinsic apoptotic pathway (caspase-8 cleavage) was found. Finally, a mitochondrial localization of SLC4A7 was demonstrated. The electroneutral sodium bicarbonate co-transporter SLC4A7 localizes in mitochondria and suppresses the ischaemia-induced activation of the mitochondrial pathway of apoptosis in coronary EC.

Live-imaging analysis of apoptosis and caspase activation reveals that apoptosis is a facilitator of the neural tube closure

Live-imaging analysis of apoptosis and caspase activation reveals that apoptosis is a facilitator of the neural tube closure