Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X₇ receptors (P2X₇Rs). However, a link between P2X₇Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4⁺) and cytotoxic (CD8⁺) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (¹⁰Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1^?/? mice. Moreover, electrophysiological measurements in wild-type CD4⁺ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1^?/? mice, in which levels of P2X₇Rs and ATP-induced intracellular free Ca²⁺ responses were enhanced suggesting that P2X₇Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.     

Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

Urothelial cells respond to bladder distension with ATP release, and ATP signaling within the bladder and from the bladder to the CNS is essential for proper bladder function. In other cell types, pannexin 1 (Panx1) channels provide a pathway for mechanically-induced ATP efflux and for ATP-induced ATP release through interaction with P2X₇ receptors (P2X₇Rs). We report that Panx1 and P2X₇R are functionally expressed in the bladder mucosa and in immortalized human urothelial cells (TRT-HU1), and participate in urothelial ATP release and signaling. ATP release from isolated rat bladders induced by distention was reduced by the Panx1 channel blocker mefloquine (MFQ) and was blunted in mice lacking Panx1 or P2X₇R expression. Hypoosmotic shock induced YoPro dye uptake was inhibited by MFQ and the P2X₇R blocker A438079 in TRT-HU1 cells, and was also blunted in primary urothelial cells derived from mice lacking Panx1 or P2X₇R expression. Rinsing-induced mechanical stimulation of TRT-HU1 cells triggered ATP release, which was reduced by MFQ and potentiated in low divalent cation solution (LDPBS), a condition known to enhance P2X₇R activation. ATP signaling evaluated as intercellular Ca²⁺ wave radius was significantly larger in LDPBS, reduced by MFQ and by apyrase (ATP scavenger). These findings indicate that Panx1 participates in urothelial mechanotransduction and signaling by providing a direct pathway for mechanically-induced ATP release and by functionally interacting with P2X₇Rs.     

Inhibition of drug‐induced Fas ligand transcription and apoptosis by Bcl‐XL

Fas/Fas ligand system triggers apoptosis in many cell types. Bcl‐X_L overexpresion antagonizes Fas/Fas ligand‐mediated cell death. The mechanism by which Bcl-X_L influences Fas‐mediated cell death is unclear. We have found that microtubule‐damaging drugs (e.g. Paclitaxel) induce apoptosis in a Fas/FasL‐dependent manner. Inhibition of Fas/FasL pathway by anti‐FasL antibody, mutant Fas or a dominant negative FADD blocks paclitaxel‐induced apoptosis. Paclitaxel induced apoptosis through activation of both caspase‐8 and caspase‐3. Overexpression of Bcl‐X_L leads to inhibition of paclitaxel‐induced FasL expression and apoptosis. Bcl‐X_L prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes) by inhibiting the activation of calcineurin, a calcium‐dependent phosphatase that must dephosphorylate NFAT for it to move to the nucleus. The loop domain in Bcl‐X_L can suppress the anti‐apoptotic function of Bcl‐X_L and may be a target for regulatory post‐translational modifications. Upon phosphorylation, Bcl‐X_L loses its ability to bind with calcineurin. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, paclitaxel and other drugs that disturb microtubule function kill cells, at least in part, through the induction of FasL, and Bcl‐X_L‐mediated resistance to these agents is related to failure to induce FasL expression.     

Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X₇ receptors (P2X₇Rs). However, a link between P2X₇Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4⁺) and cytotoxic (CD8⁺) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (¹⁰Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1^−/− mice. Moreover, electrophysiological measurements in wild-type CD4⁺ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1^−/− mice, in which levels of P2X₇Rs and ATP-induced intracellular free Ca²⁺ responses were enhanced suggesting that P2X₇Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.     

Taiwan cobra phospholipase A2‐elicited JNK activation is responsible for autocrine fas‐mediated cell death and modulating Bcl‐2 and Bax protein expression in human leukemia K562 cells

Phospholipase A₂ (PLA₂) from Naja naja atra venom induced apoptotic death of human leukemia K562 cells. Degradation of procaspases, production of tBid, loss of mitochondrial membrane potential, Bcl‐2 degradation, mitochondrial translocation of Bax, and cytochrome c release were observed in PLA₂‐treated cells. Moreover, PLA₂ treatment increased Fas and FasL protein expression. Upon exposure to PLA₂, activation of p38 MAPK (mitogen‐activated protein kinase) and JNK (c‐Jun NH₂‐terminal kinase) was found in K562 cells. SB202190 (p38 MAPK inhibitor) pretreatment enhanced cytotoxic effect of PLA₂ and led to prolonged JNK activation, but failed to affect PLA₂‐induced upregulation of Fas and FasL protein expression. Sustained JNK activation aggravated caspase8/mitochondria‐dependent death pathway, downregulated Bcl‐2 expression and increased mitochondrial translocation of Bax. SP600125 (JNK inhibitor) abolished the cytotoxic effect of PLA₂ and PLA₂‐induced autocrine Fas death pathway. Transfection ASK1 siRNA and overexpression of dominant negative p38α MAPK proved that ASK1 pathway was responsible for PLA₂‐induced p38 MAPK and JNK activation and p38α MAPK activation suppressed dynamically persistent JNK activation. Downregulation of FADD abolished PLA₂‐induced procaspase‐8 degradation and rescued viability of PLA₂‐treated cells. Taken together, our results indicate that JNK‐mediated autocrine Fas/FasL apoptotic mechanism and modulation of Bcl‐2 family proteins are involved in PLA₂‐induced death of K562 cells. J. Cell. Biochem. 109: 245–254, 2010. © 2009 Wiley‐Liss, Inc.     

Caspase‐3‐mediated cyclic stretch‐induced myoblast apoptosis via a Fas/FasL‐independent signaling pathway during myogenesis

Skeletal muscle cells are exposed to mechanical stretch during embryogenesis. Increased stretch may contribute to cell death, and the molecular regulation by stretch remains incompletely understood. The aim of this study was to investigate the effects of cyclic stretch on cell death and apoptosis in myoblast using a Flexercell Strain Unit. Apoptosis was studied by annexin V binding and PI staining, DNA size analysis, electron microphotograph, and caspase assays. Fas/FasL expression was determined by Western blot. When myoblasts were cultured on a flexible membrane and subjected to cyclic strain stress, apoptosis was observed in a time‐dependent manner. We also determined that stretch induced cleavage of caspase‐3 and increased caspase‐3 activity. Caspase‐3 inhibition reduced stretch‐induced apoptosis. Protein levels of Fas and FasL remained unchanged. Our findings implicated that stretch‐induced cell death is an apoptotic event, and that the activation of caspase cascades is required in stretch‐induced cell apoptosis. Furthermore, we had provided evidence that caspase‐3 mediated cyclic stretch‐induced myoblast apoptosis. Mechanical forces induced activation of caspase‐3 via signaling pathways independent of Fas/FasL system. J. Cell. Biochem. 107: 834–844, 2009. © 2009 Wiley‐Liss, Inc.     

Upregulation of Fas and FasL in Taiwan cobra phospholipase A2‐treated human neuroblastoma SK‐N‐SH cells through ROS‐ and Ca2+‐mediated p38 MAPK activation

The aim of the present study is to elucidate the signaling pathway involved in death of human neuroblastoma SK‐N‐SH cells induced by Naja naja atra phospholipase A₂ (PLA₂). Upon exposure to PLA₂, p38 MAPK activation, ERK inactivation, ROS generation, increase in intracellular Ca²⁺ concentration, and upregulation of Fas and FasL were found in SK‐N‐SH cells. SB202190 (p38MAPK inhibitor) suppressed upregulation of Fas and FasL. N‐Acetylcysteine (ROS scavenger) and BAPTA‐AM (Ca²⁺ chelator) abrogated p38 MAPK activation and upregulation of Fas and FasL expression, but restored phosphorylation of ERK. Activated ERK was found to attenuate p38 MAPK‐mediated upregulation of Fas and FasL. Deprivation of catalytic activity could not diminish PLA₂‐induced cell death and Fas/FasL upregulation. Moreover, the cytotoxicity of arachidonic acid and lysophosphatidylcholine was not related to the expression of Fas and FasL. Taken together, our results indicate that PLA₂‐induced cell death is, in part, elicited by upregulation of Fas and FasL, which is regulated by Ca²⁺‐ and ROS‐evoked p38 MAPK activation, and suggest that non‐catalytic PLA₂ plays a role for the signaling pathway. J. Cell. Biochem. 106: 93–102, 2009. © 2008 Wiley‐Liss, Inc.     

Suppression of ERK signaling evokes autocrine Fas‐mediated death in arachidonic acid‐treated human chronic myeloid leukemia K562 cells

Arachidonic acid (AA)‐induced apoptotic death of K562 cells (human chronic myeloid leukemic cells) was characteristic of reactive oxygen species (ROS) generation and mitochondrial depolarization. N‐Acetylcysteine pretreatment rescued viability of AA‐treated cells and abolished mitochondrial depolarization. In contrast to no significant changes in phospho‐JNK and phospho‐ERK levels, AA evoked notable activation of p38 MAPK. Unlike that of JNK and p38 MAPK, ERK suppression further reduced the viability of AA‐treated cells. Increases in Fas/FasL protein expression, caspase‐8 activation, the production of tBid and the loss of mitochondrial membrane potential were noted with K562 cells that were treated with a combination of U0126 and AA. Down‐regulation of FADD attenuated U0126‐evoked degradation of procaspase‐8 and Bid. Abolition of p38 MAPK activation abrogated U0126‐elicited Fas/FasL up‐regulation in AA‐treated cells. U0126 pretreatment suppressed c‐Fos phosphorylation but increased p38 MAPK‐mediated c‐Jun phosphorylation. Knock‐down of c‐Fos and c‐Jun protein expression by siRNA suggested that c‐Fos counteracted the effect of c‐Jun on Fas/FasL up‐regulation. Taken together, our data indicate that AA induces the ROS/mitochondria‐dependent death pathway and blocks the ERK pathway which enhances the cytotoxicity of AA through additionally evoking an autocrine Fas‐mediated apoptotic mechanism in K562 cells. J. Cell. Physiol. 222: 625–634, 2010. © 2009 Wiley‐Liss, Inc.     

P2X7 nucleotide receptors mediate caspase-8/9/3-dependent apoptosis in rat primary cortical neurons

Apoptosis is a major cause of cell death in the nervous system. It plays a role in embryonic and early postnatal brain development and contributes to the pathology of neurodegenerative diseases. Here, we report that activation of the P2X₇ nucleotide receptor (P2X₇R) in rat primary cortical neurons (rPCNs) causes biochemical (i.e., caspase activation) and morphological (i.e., nuclear condensation and DNA fragmentation) changes characteristic of apoptotic cell death. Caspase-3 activation and DNA fragmentation in rPCNs induced by the P2X₇R agonist BzATP were inhibited by the P2X₇R antagonist oxidized ATP (oATP) or by pre-treatment of cells with P2X₇R antisense oligonucleotide indicating a direct involvement of the P2X₇R in nucleotide-induced neuronal cell death. Moreover, Z-DEVD-FMK, a specific and irreversible cell permeable inhibitor of caspase-3, prevented BzATP-induced apoptosis in rPCNs. In addition, a specific caspase-8 inhibitor, Ac-IETD-CHO, significantly attenuated BzATP-induced caspase-9 and caspase-3 activation, suggesting that P2X₇R-mediated apoptosis in rPCNs occurs primarily through an intrinsic caspase-8/9/3 activation pathway. BzATP also induced the activation of C-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated kinases (ERK1/2) in rPCNs, and pharmacological inhibition of either JNK1 or ERK1/2 significantly reduced caspase activation by BzATP. Taken together, these data indicate that extracellular nucleotides mediate neuronal apoptosis through activation of P2X₇Rs and their downstream signaling pathways involving JNK1, ERK and caspases 8/9/3.     

Cell death induced by the Fas/Fas ligand pathway and its role in pathology.

Engagement of the cell death surface receptor Fas by Fas ligand (FasL) results in apoptotic cell death, mediated by caspase activation. Cell death mediated via Fas/FasL interaction is important for homeostasis of cells in the immune system and for maintaining immune-privileged sites in the body. Killing via the Fas/FasL pathway also constitutes an important pathway of killing for cytotoxic T cells. Fas ligand is induced in activated T cells, resulting in activation-induced cell death by the Fas/FasL pathway. Recently it has been shown that the Fas receptor can also be up-regulated following a lesion to the cell, particularly that induced by DNA-damaging agents. This can then result in killing of the cell by a Fas/FasL-dependent pathway. Up-regulation of Fas receptor following DNA damage appears to be p53 dependent.     

Functional significance of the negative-feedback regulation of ATP release via pannexin-1 hemichannels under ischemic stress in astrocytes

The opening of pannexin-1 (Px1) hemichannels is regulated by the activity of P2X(7) receptors (P2X(7)Rs). At present, however, little is known about how extracellular ATP-sensitive P2X(7)Rs regulates the opening and closure of Px1 hemichannels. Several lines of evidence suggest that P2X(7)Rs are activated under pathological conditions such as ischemia, resulting in the opening of Px1 hemichannels responsible for the massive influx of Ca(2+) from the extracellular space and the release of ATP from the cytoplasm, leading to cell death. Here we show in cultured astrocytes that the suppression of the activity of P2X(7)Rs during simulated ischemia (oxygen/glucose deprivation, OGD) resulted in the opening of Px1 hemichannels, leading to the enhanced release of ATP. In addition, the suppression of the activity of P2X(7)Rs during OGD resulted in a significant increase in astrocytic damage. Both the P2X(7)Rs suppression-induced enhancement of the release of ATP and cell damage were reversed by co-treatment with blockers of Px1 hemichannels, suggesting that suppression of the activity of PX(7)Rs resulted in the opening of Px1 hemichannels. All these findings suggested the existence of a negative-feedback loop regulating the release of ATP via Px1 hemichannels; ATP-induced suppression of ATP release. The present study indicates that ATP, released through Px1 hemichannels, activates P2X(7)Rs, resulting in the closure of Px1 hemichannels during ischemia. This negative-feedback mechanism, suppressing the loss of cellular ATP and Ca(2+) influx, might contribute to the survival of astrocytes under ischemic stress.     

Connexin43 and pannexin1 channels in osteoblasts: who is the "hemichannel"?

Osteoblasts sense and respond to mechanical stimuli in a process involving influx and release of large ions and signaling molecules. Unapposed gap junction hemichannels formed of connexin43 (Cx43) have been proposed as a major route for such exchange, in particular for release of ATP and prostaglandin E₂ (PGE₂) in osteocytes. However, we have found that Cx43-null osteoblasts have unaltered, mechanically induced PGE₂ release and ATP-induced YoPro dye uptake. In contrast, PGE₂ release in response to fluid shear stress is abolished in P2X₇ receptor (P2X₇R)–null osteoblasts, and ATP-induced dye uptake is attenuated following treatment of wild-type cells with a P2X₇R or Pannexin1 (Panx1) channel blocker. These data indicate that Panx1 channels, in concert with P2X₇R, likely form a molecular complex that performs the hemichannel function in osteoblast mechanosignaling.     

Connexin hemichannel and pannexin channel electrophysiology: How do they differ?

Connexin hemichannels are postulated to form a cell permeabilization pore for the uptake of fluorescent dyes and release of cellular ATP. Connexin hemichannel activity is enhanced by low external [Ca²⁺]_o, membrane depolarization, metabolic inhibition, and some disease-causing gain-of-function connexin mutations. This paper briefly reviews the electrophysiological channel conductance, permeability, and pharmacology properties of connexin hemichannels, pannexin 1 channels, and purinergic P2X₇ receptor channels as studied in exogenous expression systems including Xenopus oocytes and mammalian cell lines such as HEK293 cells. Overlapping pharmacological inhibitory and channel conductance and permeability profiles makes distinguishing between these channel types sometimes difficult. Selective pharmacology for Cx43 hemichannels (Gap19 peptide), probenecid or FD&C Blue #1 (Brilliant Blue FCF, BB FCF) for Panx1, and A740003, A438079, or oxidized ATP (oATP) for P2X7 channels may be the best way to distinguish between these three cell permeabilizing channel types. Endogenous connexin, pannexin, and P2X₇ expression should be considered when performing exogenous cellular expression channel studies. Cell pair electrophysiological assays permit the relative assessment of the connexin hemichannel/gap junction channel ratio not often considered when performing isolated cell hemichannel studies.     

P2X7 receptor‐induced death of motor neurons by a peroxynitrite/FAS‐dependent pathway

The P2X7 receptor/channel responds to extracellular ATP and is associated with neuronal death and neuroinflammation in spinal cord injury and amyotrophic lateral sclerosis. Whether activation of P2X7 directly causes motor neuron death is unknown. We found that cultured motor neurons isolated from embryonic rat spinal cord express P2X7 and underwent caspase‐dependent apoptosis when exposed to exceptionally low concentrations of the P2X7 agonist 2′(3′)‐O‐(4‐Benzoylbenzoyl)‐ATP. The P2X7 inhibitors BBG, oATP, and KN‐62 prevented 2′(3′)‐O‐(4‐Benzoylbenzoyl)‐ATP‐induced motor neuron death. The endogenous P2X7 agonist ATP induced motor neuron death at low concentrations (1‐100 μM). High concentrations of ATP (1 mM) paradoxically became protective due to degradation in the culture media to produce adenosine and activate adenosine receptors. P2X7‐induced motor neuron death was dependent on neuronal nitric oxide synthase‐mediated production of peroxynitrite, p38 activation, and autocrine FAS signaling. Taken together, our results indicate that motor neurons are highly sensitive to P2X7 activation, which triggers apoptosis by activation of the well‐established peroxynitrite/FAS death pathway in motor neurons.     

Fas/FasL-dependent and -independent activation of caspase-8 in doxorubicin-treated human breast cancer MCF-7 cells: ADAM10 down-regulation activates Fas/FasL signaling pathway

The contribution of Fas-mediated death pathway to doxorubicin-induced death of MCF-7 cells is not unambiguously elucidated. Thus, this study was conducted to explore doxorubicin-induced Fas/FasL signaling pathway activation in MCF-7 cells and doxorubicin-resistant MCF-7 (MCF-7/Dox) cells. Doxorubicin-induced caspase-8 activation was found to be mediated through Akt/ERK inactivation and FasL-independent Fas pathway in MCF-7 cells, while caspase-8 activation in MCF-7/Dox cells depended exclusively on FasL-stimulated Fas pathway. Suppression of caspase-8 activation restored the viability of doxorubicin-treated MCF-7 cells and MCF-7/Dox cells. Contrary to FasL surface expression exclusively detected in MCF-7/Dox cells, intracellular FasL expression was noted with MCF-7 cells. Promotion of FasL translocation to the cell surface by lysophosphatidic acid evoked a FasL-activated Fas death pathway in MCF-7 cells. Doxorubixin-evoked β-TrCP up-regulation promoted Sp1 degradation, which subsequently suppressed ADAM10 expression in MCF-7 and MCF-7/Dox cells. Doxorubicin-induced down-regulation of ADAM10 reduced FasL shedding, leading to Fas pathway activation in MCF-7/Dox cells. Knock-down of ADAM10 induced death in MCF-7/Dox cells, but marginally reduced the viability of MCF-7 cells. Taken together, our data indicate that Akt/ERK-mediated caspase-8 activation and Fas/FasL-mediated caspase-8 activation mostly elucidate doxorubicin-induced death in MCF-7 cells and MCF-7/Dox cells, respectively. These observations suggest a promising therapeutic modality for overcoming doxorubicin-resistant breast cancer by targeting ADAM10 sheddase activity.     

Adenosine‐5′‐triphosphate suppresses proliferation and migration capacity of human endometrial stem cells

Extracellular ATP through the activation of the P2X and P2Y purinergic receptors affects the migration, proliferation and differentiation of many types of cells, including stem cells. High plasticity, low immunogenicity and immunomodulation ability of mesenchymal stem cells derived from human endometrium (eMSCs) allow them to be considered a prominent tool for regenerative medicine. Here, we examined the role of ATP in the proliferation and migration of human eMSCs. Using a wound healing assay, we showed that ATP‐induced activation of purinergic receptors suppressed the migration ability of eMSCs. We found the expression of one of the ATP receptors, the P2X₇ receptor in eMSCs. In spite of this, cell activation with specific P2X₇ receptor agonist, BzATP did not significantly affect the cell migration. The allosteric P2X₇ receptor inhibitor, AZ10606120 also did not prevent ATP‐induced inhibition of cell migration, confirming that inhibition occurs without P2X₇ receptor involvement. Flow cytometry analysis showed that high concentrations of ATP did not have a cytotoxic effect on eMSCs. At the same time, ATP induced the cell cycle arrest, suppressed the proliferative and migration capacity of eMSCs and therefore could affect the regenerative potential of these cells.     

Swainsonine Induces Caprine Luteal Cells Apoptosis via Mitochondrial‐Mediated Caspase‐Dependent Pathway

Swainsonine (SW) is an indolizidine alkaloid isolated from a number of poisonous plants. We have previously reported that SW inhibited luteal cell progesterone production by inducing caprine luteal cell apoptosis in vitro; however, the molecular mechanism of this phenomenon remains unclear. In this study, SW‐treated luteal cells showed apoptosis characteristics, including nuclear fragmentation, DNA ladder formation, and phosphatidylserine externalization. Further studies showed that SW activated caspase‐9 and caspase‐3, which subsequently cleaved poly(ADP‐ribose) polymerase. SW also increased in Bax/BcL‐2 ratios, promoted Bax translocation from the cytosol to mitochondria, and triggered the release of cytochrome c from mitochondria into the cytoplasm. However, Fas and Fas ligand induction or caspase‐8 activity did not appear any significant changes. Additional analysis also showed that pan‐caspase inhibitor, caspase‐9 inhibitor, or caspase‐3 inhibitor almost completely protected the cells from SW‐induced apoptosis, but not caspase‐8 inhibitor. Overall, these data demonstrated that SW induced luteal cells apoptosis through a mitochondrial‐mediated caspase‐dependent pathway.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Akt‐mediated anti‐apoptotic effects of substance P in Anti‐Fas‐induced apoptosis of human tenocytes

Substance P (SP) and its receptor, the neurokinin‐1 receptor (NK‐1 R), are expressed by human tenocytes, and they are both up‐regulated in cases of tendinosis, a condition associated with excessive apoptosis. It is known that SP can phosphorylate/activate the protein kinase Akt, which has anti‐apoptotic effects. This mechanism has not been studied for tenocytes. The aims of this study were to investigate if Anti‐Fas treatment is a good apoptosis model for human tenocytes in vitro, if SP protects from Anti‐Fas‐induced apoptosis, and by which mechanisms SP mediates an anti‐apoptotic response. Anti‐Fas treatment resulted in a time‐ and dose‐dependent release of lactate dehydrogenase (LDH), i.e. induction of cell death, and SP dose‐dependently reduced the Anti‐Fas‐induced cell death through a NK‐1 R specific pathway. The same trend was seen for the TUNEL assay, i.e. SP reduced Anti‐Fas‐induced apoptosis via NK‐1 R. In addition, it was shown that SP reduces Anti‐Fas‐induced decrease in cell viability as shown with crystal violet assay. Protein analysis using Western blot confirmed that Anti‐Fas induces cleavage/activation of caspase‐3 and cleavage of PARP; both of which were inhibited by SP via NK‐1 R. Finally, SP treatment resulted in phosphorylation/activation of Akt as shown with Western blot, and it was confirmed that the anti‐apoptotic effect of SP was, at least partly, induced through the Akt‐dependent pathway. In conclusion, we show that SP reduces Anti‐Fas‐induced apoptosis in human tenocytes and that this anti‐apoptotic effect of SP is mediated through NK‐1 R and Akt‐specific pathways.     

Non‐hemolytic enterotoxin of Bacillus cereus induces apoptosis in Vero cells

Bacillus cereus is an opportunistic pathogen that often causes foodborne infectious diseases and food poisoning. Non‐hemolytic enterotoxin (Nhe) is the major toxin found in almost all enteropathogenic B. cereus and B. thuringiensis isolates. However, little is known about the cellular response after Nhe triggered pore formation on cell membrane. Here, we demonstrate that Nhe induced cell cycle arrest at G₀/G₁ phase and provoked apoptosis in Vero cells, most likely associated with mitogen‐activated protein kinase (MAPK) and death receptor pathways. The influx of extracellular calcium ions and increased level of reactive oxygen species in cytoplasm were sensed by apoptosis signal‐regulating kinase 1 (ASK1) and p38 MAPK. Extrinsic death receptor Fas could also promote the activation of p38 MAPK. Subsequently, ASK1 and p38 MAPK triggered downstream caspase‐8 and 3 to initiate apoptosis. Our results clearly demonstrate that ASK1, and Fas‐p38 MAPK‐mediated caspase‐8 dependent pathways are involved in apoptotic cell death provoked by the pore‐forming enterotoxin Nhe.