A Potential Association between Helicobacter pylori CagA EPIYA and Multimerization Motifs with Cytokeratin 18 Cleavage Rate during Early Apoptosis

Background and Aims: Helicobacter pylori is a highly diverse pathogen, which encounters epithelial cells as the initial defense barrier during its lifelong infection. The structure of epithelial cells can be disrupted through cleavage of microfilaments. Cytokeratin 18 (CK18) is an intermediate filament, the cleavage of which is considered an early event during apoptosis following activation of effector caspases. Methods: Helicobacter pylori strains were isolated from 76 dyspeptic patients. cagA 3’ variable region and CagA protein status were analyzed by PCR and western blotting, respectively. Eight hours post‐co‐culture of AGS cells with different H. pylori strains, flow cytometric analysis was performed using M30 monoclonal antibody specific to CK18 cleavage‐induced neo‐epitope. Results: Higher rates of CK18 cleavage were detected during co‐culture of AGS cells with H. pylori strains bearing greater numbers of cagA EPIYA‐C and multimerization (CM) motifs. On the other hand, H. pylori strains with greater numbers of EPIYA‐B relative to EPIYA‐C demonstrated a decrease in CK18 cleavage rate. Thus, H. pylori‐mediated cleavage of CK18 appeared proportional to the number of CagA EPIYA‐C and CM motifs, which seemed to be downplayed in the presence of EPIYA‐B motifs. Conclusions: Our observation associating the heterogeneity of cagA variants with the potential of H. pylori strains in the induction of CK18 cleavage as an early indication of apoptosis in gastric epithelial cells supports the fact that apoptosis may be a type‐specific trait. However, additional cagA‐targeted experiments are required to clearly identify the role of EPIYA and CM motifs in apoptosis and/or the responsible effector molecules.     

DEDD association with cytokeratin filaments correlates with sensitivity to apoptosis

The cytokeratin 8/18 (CK8/18) cytoskeleton network is an early target for caspase cleavage during apoptosis. Recent reports suggest that the highly conserved and ubiquitous death effector domain containing DNA binding protein (DEDD) plays a role in the recruitment of procaspase-9 and -3 at this CK8/18 scaffold. DEDD interacts with both the CK8/18 intermediate filament network and procaspase-3 and –9. It is suggested that the CK8/18 fibrils may provide a scaffold for the proximity-induced autocleavage and activation of procaspase-9 in close association with caspase-3. We addressed this issue by investigating DEDD staining patterns in various cell lines and by correlating these expression patterns with the sensitivity of these cell lines for roscovitine-induced apoptosis. We showed that in some cell lines DEDD revealed a bright filamentous staining pattern in others DEDD staining was weak and diffusely distributed in the cytoplasm of the cells. The difference in staining patterns was irrespective of the phosphorylation status of the cytokeratin filaments. In cells showing a filamentous staining pattern, DEDD was strongly associated with the CK8/18 cytokeratin filaments as evidenced by double immunofluorescence and its resistance to extraction with Triton X-100. Subcellular fractionation indicates that DEDD co-purifies with CK18, which corroborates a strong association of DEDD and the cytokeratin network. DEDD was either mono- or diubiquinated. Cells showing a filamentous DEDD distribution are more apoptosis-prone as evidenced by the rapid appearance of M30 CytoDeath-positive cells after induction of apoptosis. The sensitivity towards apoptosis is irrespective of the procaspase-3 content of the cells. Our data support the notion that DEDD-mediated accumulation of procaspases at the cytokeratin scaffold leads to an increase in the local concentration, which renders cells more apoptosis-prone.     

Advances in cytochemical methods for detection of apoptosis.

In an earlier article from this laboratory, the current methods developed to detect apoptosis in cells and tissues were highlighted, along with the challenges in their interpretation. Recent discoveries concerning the underlying biochemical mechanisms of apoptotic effector pathways have made possible further assays that allow a more direct measure of the activation of the apoptotic machinery in cells. This article summarizes some of these newer methods and extends the interpretation of the more classical assays of apoptosis in a defined cell system. We present data in KB and PC3 cell model culture systems induced to undergo apoptosis by the plant toxin ricin. Using a modified in situ nick translation assay (ISNT) with either Bodipy or BUdR labeling, we confirm that most cells showing altered nuclear morphology do not show reactivity with this assay until very late in the apoptotic process. We also show that only a minority of cells label with fluorescent annexin V during apoptosis but that apoptotic cells continue to internalize material from the cell surface through endocytosis after becoming reactive with annexin V. In addition, we describe the utility of a prototype of new assays for caspase substrate cleavage products, the detection of cleaved cytokeratin 18. It is these newer cleavage product assays that perhaps hold the greatest promise for specific detection of apoptosis in cells either in cell culture or in intact tissues. (J Histochem Cytochem 49:821-832, 2001)     

Induction of apoptosis by antisense CK2 in human prostate cancer xenograft model

Protein serine/threonine kinase CK2 (formerly casein kinase 2) is a ubiquitous protein kinase that plays key roles in cell growth, proliferation, and survival. We have shown previously that its molecular down-regulation induces apoptosis in cancer cells in culture. Here, we have employed a xenograft model of prostate cancer to extend these studies to determine whether antisense CK2alpha evokes a similar response in vivo. A single dose of antisense CK2alpha oligodeoxynucleotide given directly into the PC3-LN4 xenograft tumor in nude mouse induced a dose- and time-dependent tumor cell death in vivo. The tumor was completely resolved at the higher tested dose of the antisense. Cell death was due to apoptosis and correlated with a potent down-regulation of the CK2alpha message and loss of CK2 from the nuclear matrix in the xenograft tissue as well as in cancer cells in culture. These observations accorded with several of the earlier studies indicating that loss of CK2 from the nuclear matrix is associated with induction of apoptosis. Comparison of the effects of antisense CK2alpha oligodeoxynucleotide on cancer versus normal or noncancer cells showed that the concentration of antisense CK2alpha that elicited extensive apoptosis in tumor cells in culture or xenograft tumors in vivo had a relatively small or minimal effect on noncancer cells in culture or on normal prostate gland subjected to orthotopic injection of antisense oligodeoxynucleotide in vivo. The basis for the difference in sensitivity of cancer versus noncancer cells to antisense CK2alpha is unknown at this time; however, this differential response under similar conditions of treatment may be significant in considering the potential feasibility of targeting the CK2 signal for induction of apoptosis in cancer cells in vivo. Although much further work will be needed to establish the feasibility of targeting CK2 for cancer therapy, to our knowledge, this is the first report to provide important new evidence as an initial "proof of principle" for the potential application of antisense CK2alpha in cancer therapy, paving the way for future detailed studies of approaches to targeting CK2 in vivo to induce cancer cell death.     

Downregulation of CK2 induces apoptosis in cancer cells – A potential approach to cancer therapy

We have previously documented that naked antisense CK2α ODN can potently induce apoptosis in cancer cells in culture and in mouse xenograft human prostate cancer. The effects of the antisense CK2α are related to downregulation of CK2α message and rapid loss of the CK2 from the nuclear compartment. Here we demonstrate that downregulation of CK2 elicited by diverse methods leads to inhibition of cell growth and induction of apoptosis. The various approaches to downregulation of CK2 employed were transfection with kinase-inactive plasmid, use of CK2α siRNA, use of inhibitors of CK2 activity, and use of antisense CK2α ODN packaged in sub-50 nm nanocapsules made from tenascin. In all cases, the downregulation of CK2 is associated with loss in cell survival. We have also described preliminary observations on an approach to targeting CK2 in cancer cells. For this, sub-50 nm tenascin-based nanocapsules bearing the antisense CK2α ODN were employed to test that the antisense is delivered to the cancer cells in vivo. The results provide the first preliminary evidence that such an approach may be feasible for targeting CK2 in cancer cells. Together, our results suggest that CK2 is potentially a highly plausible target for cancer therapy.     

Release of cytokeratin-18 and -19 fragments (TPS and CYFRA 21-1) into the extracellular space during apoptosis

Serum fragments of cytokeratins-18 and -19 (measured as TPS and CYFRA 21-1, respectively) have traditionally been considered as markers of tumor proliferation, although the evidence is scarce for a causative relationship between proliferation and levels of TPS and CYFRA 21-1. We examined whether apoptosis might produce TPS and CYFRA 21-1 fragments. MCF-7 breast cancer cells were treated with mitomycin C or agonistic anti-CD95 antibody, and levels of TPS and CYFRA 21-1 in tissue culture supernatants were compared with the frequency of cells exhibiting the following markers of cell death: intracellular cytokeratin-18 cleavage, surface staining with annexin-V, propidium iodide uptake, DNA fragmentation. Twenty-four hours after inducing apoptosis, levels of TPS and CYFRA 21-1 were elevated > or = 4-fold in culture supernatants. Elevations in TPS and CYFRA 21-1 coincided with apoptosis measured by the first three cell death markers but preceded DNA fragmentation. These mitomycin C- and CD95-mediated elevations were completely inhibited by co-incubation with the caspase inhibitors Z-VAD.fmk and Z-IETD.fmk, respectively. We conclude that TPS and CYFRA 21-1 can be abundantly released into the extracellular space during the intermediate stage of epithelial cell apoptosis.     

Cleavage of the Junin Virus Nucleoprotein Serves a Decoy Function To Inhibit the Induction of Apoptosis during Infection

The regulation of apoptosis during infection is an important factor for host survival and, in some cases, also for the virus life cycle. At the same time, mechanisms to prevent the induction of apoptosis have been observed in numerous viral pathogens, but until now the role of apoptosis during arenavirus infection has not been investigated. Junin virus (JUNV) belongs to the New World arenavirus serogroup of the Arenaviridae and is the causative agent of Argentine hemorrhagic fever. We have demonstrated that infection with JUNV in cell culture does not induce apoptosis but leads to cleavage of the nucleoprotein (NP) into discrete products resembling caspase cleavage events. Similar specific NP degradation patterns were also observed in NP-transfected cell lines, and a closer examination of the sequence of NP showed several putative caspase cleavage motifs. Point mutations that abolished these cleavage motifs were consistent with the loss of certain cleavage products. Consistent with these data, further studies showed that treatment with a caspase inhibitor also reduced NP cleavage, indicating that the observed cleavage events were occurring as a result of caspase activity with NP as a substrate. Finally, we showed that expression of NP suppresses the cleavage of caspase 3 in cells treated with an apoptosis activator. Based on these findings, we propose that NP functions as a decoy substrate for caspase cleavage in order to inhibit the induction of apoptosis in JUNV-infected cells.     

Apoptosis in the developing human heart resembles apoptosis in epithelial tissues

It is widely accepted that apoptosis plays an important role in the development of the heart as well as in different heart diseases. Despite extensive research efforts, many issues regarding apoptosis in the heart remain unsolved, including the detection of apoptotic cardiomyocytes, their morphological features, the mechanisms of their removal and the clinical significance of apoptosis in the heart. It has been suggested that fetal cardiomyocytes resemble epithelial tissues. To test this hypothesis, we analyzed the expression of an epithelial marker cytokeratin 18 (CK18) and caspase-cleaved-CK18, recognized by antibody M30, as well as the expression of cleaved caspase-3 and desmosomal and classical cadherins, major components of desmosomes and adherens junctions in fetal hearts in comparison to infant and adult human hearts. We found that, in fetal hearts, cardiomyocytes expressed CK18 and that apoptotic cardiomyocytes expressed caspase-cleaved CK18, being recognized by antibody M30. Furthermore, desmosomal and classical cadherins exhibited a membraneous reaction similar to epithelial tissues. In adults and children after the age of 6 months, cadherins were localized in the intercalated disks, cardiomyocytes lost CK18 expression and apoptotic cardiomyocytes were no longer recognized by M30. We conclude that apoptosis in the developing human heart resembles apoptosis in epithelial tissues, exhibiting different characteristics than in the adult human heart.     

Apoptosis enzyme-linked immunosorbent assay distinguishes anticancer drugs from toxic chemicals and predicts drug synergism

The effects of anticancer drugs and toxic compounds on leukemic cells in culture were evaluated by enzyme-linked-immunosorbent assay (ELISA) based on the detection of apoptotic cells by a monoclonal antibody against single-stranded DNA. The concentrations of 13 anticancer drugs, which increased apoptosis ELISA absorbance, were similar to the concentrations decreasing long-term cell survival. Short-term metabolic tetrazolium-based 3-(4,5-dimethylthiazol-yl)-2,5-diphenyformazan bromide (MTT) assay was significantly less sensitive than apoptosis ELISA and the cell survival assay. In contrast to anticancer drugs, 12 toxic chemicals did not increase apoptosis ELISA absorbance at cytotoxic concentrations. The difference between two groups of compounds by apoptosis ELISA was especially large in cultures treated with twofold of concentrations producing 50% inhibition of cell growth: all anticancer drugs induced intense reaction (mean absorbance 2.0), while none of the toxic chemicals induced apoptosis. The application of apoptosis ELISA to chemosensitivity testing was evaluated by its ability to detect synergism of anticancer drug combinations. Among 66 drug combinations tested, only combination of nitrogen mustard with mithramycin was highly synergistic by the apoptosis ELISA, as defined by apoptosis induction with the combination containing each drug at 50% of effective concentration. This combination was also synergistic in the cell survival assay, producing significant cell kill while each drug alone had no effect on cell survival. This synergism was not detected by MTT assay. We conclude that apoptosis ELISA could be useful for drug development and chemosensitivity assessment as it can distinguish clinically useful anticancer drugs from toxic compounds, is as sensitive as the long-term cell survival assay and can detect anticancer drug synergism by rapid evaluation of apoptosis induction.     

Downregulation of protein kinase CK2 activity facilitates tumor necrosis factor-α-mediated chondrocyte death through apoptosis and autophagy

Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF)-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA) model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence versus a cause of the degeneration in vivo.     

Garlic compounds induced calpain and intrinsic caspase cascade for apoptosis in human malignant neuroblastoma SH-SY5Y cells

Malignant (N-type) neuroblastoma continues to defy current chemotherapeutic regimens. We tested the garlic compounds diallyl sulfide (DAS) and diallyl disulfide (DADS) for induction of apoptosis in human malignant neuroblastoma SH-SY5Y cells. Viability of human primary neurons was unaffected after 24 h treatment with 50 and 100 μM DAS and 50 μM DADS but slightly affected with 100 μM DADS. Treatment with 50 and 100 μM DAS or DADS significantly decreased viability in SH-SY5Y cells. Wright staining showed morphological features of apoptosis in SH-SY5Y cells treated with 50 and 100 μM DAS or DADS for 24 h. ApopTag assay demonstrated DNA fragmentation in apoptotic cells. Apoptosis was associated with an increase in [Ca²⁺]_i, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, increase in cytosolic Smac/Diablo, and down regulation of inhibitor-of-apoptosis proteins and nuclear factor kappa B (NFκB). Activation of caspase-9 and caspase-3 indicated involvement of intrinsic pathway of apoptosis. Calpain and caspase-3 activities produced 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Also, caspase-3 activity cleaved inhibitor of caspase-activated DNase (ICAD). Results strongly suggested that the garlic compounds DAS and DADS suppressed anti-apoptotic factors and activated calpain and intrinsic caspase cascade for apoptosis in SH-SY5Y cells.     

Cleavage and degradation of Claspin during apoptosis by caspases and the proteasome

Apoptosis plays a crucial role in development and tissue homeostasis. Some key survival pathways, such as DNA damage checkpoints and DNA repair, have been described to be inactivated during apoptosis. Here, we describe the processing of the human checkpoint protein Claspin during apoptosis. We observed cleavage of Claspin into multiple fragments in vivo. In vitro cleavage with caspases 3 and 7 of various fragments of the protein, revealed cut sites near the N- and C-termini of the protein. Using mass spectrometry, we identified a novel caspase cleavage site in Claspin at Asp25. Importantly, in addition to cleavage by caspases, we observed a proteasome-dependent degradation of Claspin under apoptotic conditions, resulting in a reduction of the levels of both full-length Claspin and its cleavage products. This degradation was not dependent upon the DSGxxS phosphodegron motif required for SCF(beta-TrCP)-mediated ubiquitination of Claspin. Finally, downregulation of Claspin protein levels by short interfering RNA resulted in an increase in apoptotic induction both in the presence and absence of DNA damage. We conclude that Claspin has antiapoptotic activity and is degraded by two different pathways during apoptosis. The resulting disappearance of Claspin from the cells further promotes apoptosis.     

The Role of Ca2+ on the DADS-induced Apoptosis in Mouse–Rat Hybrid Retina Ganglion Cells (N18)

Diallyl disulfide (DADS), a component of garlic, has been shown to induce growth inhibition and apoptosis in human cancer cell types. The present studies were designed to investigate the effects of DADS on mouse–rat hybrid retina ganglion cells (N18) to better understand its effect on apoptosis and apoptosis-related genes in vitro. Cell viability, cell cycle analysis, reactive oxygen species (ROS), Ca²⁺ production, mitochondria membrane potential, apoptosis induction, associated gene expression and caspases-3 activity were examined by flow cytometric assay and/or Western blot. After 24-h treatment with DADS, a dose- and time-dependent decrease in the viability of N18 cells was observed and the approximate IC₅₀ was 27.6 μM. The decreased percentage of viable cells are associated with the production of ROS then followed by the production of Ca²⁺ which is induced by DADS. DADS induced apoptosis in N18 cells via the activation of caspase-3. DADS increased the protein levels of p53, cytochrome c and phosphated JNK within 24 h of treatment and it decreased the levels of Bcl-2 and those factors may have led to the mitochondria depolarization of N18 cells. DADS induced apoptosis were accompanied by increased levels of Ca²⁺ and decreased mitochondrial membrane potential which then led to release the cytochrome c, cleavage of pro-caspase-3. Deleted levels of Ca²⁺ by BAPTA-AM 10 μM (intracellular calcium chelator) then led to decrease DADS-induced apoptosis. Inhibition of caspase-3 activation by inhibitor (z-VAD-fmk) completely blocked DADS-induced apoptosis on N18 cells. The results indicated that oxidative stress modulates cell proliferation and Ca²⁺ modulates the cell death induced by DADS.     

Identification and evaluation of apoptotic compounds from Garcinia oligantha

Four new compounds, oliganthins A-D (1-4), and one known caged xanthone gaudichaudione H (5) were isolated from the stems of Garcinia oligantha. The structures of the new compounds were elucidated by spectroscopic evidences. All of the five compounds were evaluated for their apoptosis-inducing effects using HeLa-C3 cells which have been genetically engineered to produce a fluorescent biosensor capable of detecting caspase-3 activation. All of them induced cell apoptosis at 10 μM or lower concentrations. The apoptotic activity of oliganthins A, B and gaudichaudione H were further confirmed by detecting the cleavage of PARP, which is the substrate of activated caspase-3, in these compounds-treated cells using the method of Western blot. Moreover, the values of IC(50) were measured for all five compounds on HeLa cells using the MTT assay. Among them, gaudichaudione H had the lowest IC(50) value of 0.90 μM, while the other four new compounds had IC(50) values of 1.58, 1.52, 4.15, and 7.82 μM, respectively. These results show that gaudichaudione H has the strongest apoptosis-inducing effect and cell growth inhibition effect among these xanthones and it may have the potential to be developed into a new anticancer agent.     

The Use of a Stably Expressed FRET Biosensor for Determining the Potency of Cancer Drugs

Many cancer drugs are intended to kill cancer cells by inducing apoptosis. However, the potency assays used for measuring the bioactivity of these products are generally cell viability assays which do not distinguish between cell death and growth inhibition. Here we describe a cell-based fluorescence resonance energy transfer (FRET) biosensor designed to measure the bioactivity of apoptosis inducing cancer drugs. The biosensor contains cyan fluorescent protein (CFP) linked via caspase 3 and caspase 8 specific cleavage recognition sequences to yellow fluorescent protein (YFP). Upon caspase activation, as in the case of apoptosis induction, the linker is cleaved abolishing the cellular FRET signal. This assay closely reflects the mechanism of action of cancer drugs, in killing cancer cells and therefore can function as a potency test for different cancer drugs. We rigorously demonstrate this through characterization of a class of proteins targeting the death receptors. The one-step assay appears to be superior to other apoptosis-based assays because of its simplicity, convenience, and robustness.     

Cell membrane dynamics and the induction of apoptosis by lipid compounds

To investigate the induction of apoptosis by some lipid compounds which are a potent inducer of apoptosis, the plasma membrane fluidity of U937 cells was measured using the fluorescent probe, pyrene. The increase of the membrane fluidity was observed immediately after the treatment of cells with lipid inducers. We also found that the trigger of apoptosis was pulled within 30 min after treatment. Data from the dynamic light scattering experiment indicated that lipid inducers were dissolved to form the emulsion. At the very early stage of apoptosis, possibly, the well-controlled transfer of lipid inducers from the emulsion to the lipid layer of cells can bring about the increase of membrane dynamics which might lead to the induction of apoptosis.     

Cytokeratin 18 is a specific marker of bovine intestinal M cell

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches have an important role in mucosal immune responses. A primary difficulty for investigations of bovine M cells is the lack of a specific molecular marker. To identify such a marker, we investigated the expression of several kinds of intermediate filament proteins using calf Peyer's patches. The expression patterns of cytokeratin (CK) 18 in jejunal and ileal FAE were very similar to the localization pattern of M cells recognized by scanning electron microscopy. Mirror sections revealed that jejunal CK18-positive cells had irregular and sparse microvilli, as well as pocket-like structures containing lymphocytes, typical morphological characteristic of M cells. However, CK18-negative cells had regular and dense microvilli on their surface, typical of the morphology of enterocytes. In contrast, CK20 immunoreactivity was detected in almost all villous epithelial cells and CK18-negative cells in the FAE. CK18-positive proliferating transit-amplifying cells in the crypt exchanged CK18 for CK20 above the mouth of the crypt and after moving to the villi; however, CK18-positive M cells in the crypt continued their expression of CK18 during movement to the FAE region. Terminal deoxynucleotidyl-transferase-mediated deoxyuridine-triphosphate-biotin nick-end labeling-positive apoptotic cells were specifically detected at the apical region of villi and FAE in the jejunum and ileum, and all were also stained for CK20. These data indicate that CK18 may be a molecular marker for bovine M cells in FAE and that M cells may transdifferentiate to CK20-positive enterocytes and die by apoptosis in the apex of the FAE.     

Detection of elevated caspase activation and early apoptosis in liver diseases

Apoptosis has been implicated in the pathogenesis of many diseases including various forms of liver failure. The apoptotic process is essentially regulated by intracellular proteases, called caspases, which cleave several vital proteins. Despite the rapid elucidation of apoptotic signaling cascades, however, almost no information exists about the activation of caspases in situ. In the present study, a monoclonal antibody was employed which selectively recognized cleavage site-specific fragments of the caspase substrate cytokeratin-18. We demonstrate that this antibody labeled apoptotic hepatocytes in culture and, in addition, could be used to monitor caspase activation in formalin-fixed tissue biopsies. In liver sections of different liver diseases an increased number of early apoptotic cells was detected which were not found in normal tissue. Our data reveal that hepatobiliary diseases are characterized by elevated caspase activation and apoptosis, which can be specifically detected in situ by a cleavage site-specific antibody against cytokeratin-18.     

A FRET-based microplate assay for human protein kinase CK2, a target in neoplastic disease

Besides cardiovascular diseases, cancer represents the major cause of death in developed countries. In many different human tumors, increased activity of serine/threonine protein kinase CK2 has been detected, and recent in vivo studies support a direct involvement of CK2 in tumor progression. Therefore, potent compounds to decrease CK2 activity to a non-pathogenic level would be a promising effort toward an antineoplastic therapy. In this study, an alternative to the established radiometric phosphorylation assay for quantification of CK2 activity was developed. For this purpose, the substrate peptide RRRDDDSDDD was coupled at the C-terminus to the fluorophore EDANS (5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid) and at the N-terminus to the quencher DABCYL (4-(4-dimethylaminophenylazo)benzoic acid). This resulted in quenched fluorescence of EDANS due to a FRET-based effect. After proteolytic cleavage of the peptide by elastase, the quenching effect was reduced and, as a consequence, fluorescence was increased. Because elastase is supposed to cleave at the S/D site of the peptide, phosphorylation of serine by CK2 hampered substrate binding of elastase and blocked the increase in fluorescence by proteolytic cleavage. This means that the new assay to quantify human CK2 activity is based on the differential accessibility of the proteolytic cleavage site, which is dependent on kinase phosphorylation. It could be used to measure inhibition of the human target in neoplastic diseases by the compounds TBB (4,5,6,7-tetrabromobenzotriazole) and Emodin.     

Caspase-dependent apoptosis induced by two synthetic halogenated flavanones, 3′,7-dichloroflavanone and 3′,6-dichloroflavanone,on human breast and prostate cancer cells

The destruction of cancer cells with chemotherapeutic agents is normally achieved through apoptosis. We previously introduced two synthetic halogenated flavanone derivatives, 3^′,7-dichloroflavanone (3′-7 DCF) and 3^′,6-dichloroflavanone (3′-6 DCF), as potential apoptosis-inducing agents. In the current study, we investigated the ability of these compounds in triggering intrinsic or/and extrinsic pathway of apoptosis in breast and prostate cancer cells. Also, the synergistic effect of 3′-7 DCF with TLR3 (Toll-like receptor 3) agonist in apoptosis induction was evaluated on PC3 and LNCaP human prostate cancer cells. The involved pathway of apoptosis in the treated cells was delineated by caspase-3 activity assay, PARP-1 (poly(ADP-ribose)polymerase-1) cleavage, and procaspase-9 cleavage as markers of the intrinsic pathway and procaspase-8 cleavage as the marker of the extrinsic pathway. With the exception of the normal cells, treatment of all cell lines with both 3′-7 DCF and 3′-6 DCF triggered the cleavage of procaspase-8 and procaspase-9. These results indicate that the intrinsic and the extrinsic pathways of apoptosis are the mechanisms of the toxicity of flavanones in these cancer cell lines. However, the cytoxicity of the compound 3′-7 DCF was not synergistic with TLR3 agonist. Interestingly, the activation of caspases-9 preceeded that of caspase-8 suggesting that the intrinsic pathway is the primary reason for apoptosis induction by the flavanones.