Transglutaminase 2 promotes both caspase-dependent and caspase-independent apoptotic cell death via the calpain/Bax protein signaling pathway

Transglutaminase 2 (TG2) is a versatile protein that is implicated in significant biological processes, including cell death and degenerative diseases. A possible role of TG2 in the apoptotic death of cancer cells induced by photodynamic therapy (PDT) was suggested recently; however, the mechanism by which TG2 regulates apoptotic responses to PDT remains to be elucidated. In this study, we investigated the key signaling pathways stimulated during apoptotic cell death following PDT and whether inhibition of TG2 activation using pharmacological approaches and siRNAs affects the signaling pathways. PDT caused the release of both cytochrome c and apoptosis-inducing factor (AIF) by damaging mitochondria, which resulted in caspase-dependent and caspase-independent apoptotic cell death, respectively. Released AIF translocated to the nucleus and, synergistically with the caspase-dependent pathway, led to apoptotic cell death. Both the caspase cascade and the activation of AIF following PDT were mediated by TG2 activation. In addition, PDT-activated calpain was responsible for the sequential events of Bax translocation, the collapse of ΔΨ(m), caspase-3 activation, and AIF translocation, all of which were provoked by TG2 activation. Together, these results demonstrate that PDT with a chlorin-based photosensitizer targets TG2 by activating calpain-induced Bax translocation, which induces apoptotic cell death through both caspase-dependent and AIF-mediated pathways. Moreover, these results indicate that TG2 may be a possible therapeutic target for PDT treatment of cancer.     

Subcellular localization of Photofrin determines the death phenotype of human epidermoid carcinoma A431 cells triggered by photodynamic therapy: when plasma membranes are the main targets

Photodynamic therapy (PDT) is a kind of photochemo-therapeutic treatment that exerts its effect mainly through the induction of cell death. Distinct types of cell death may be elicited by different PDT regimes. In this study, the mechanisms involved in the death of human epidermoid carcinoma A431 cells triggered by PDT with Photofrin (a clinically approved photosensitizer) were characterized. Photofrin distributes dynamically in A431 cells; the plasma membranes and Golgi complex are the main target sites of Photofrin after a brief (3 h) and prolonged (24 h) incubation, respectively. Cells with differentially localized Photofrin displayed distinct death phenotypes in response to PDT. The effects of PDT on cells with plasma membrane-localized Photofrin were further studied in details. Cells stopped proliferating post PDT at Photofrin dose >7 micro g/ml, and at higher dose (28 micro g/ml) plasma membrane disruption and cell swelling were observed immediately after PDT. Dramatic alterations of several important signaling events were detected in A431 cells post Photofrin-PDT, including (i) immediate formation of reactive oxygen species (ROS), (ii) rapid activation of c-Jun N-terminal kinase, (iii) delayed activation of caspase-3 and cleavage of polyADP-ribose polymerase and p21-activated kinase 2, and (iv) loss of mitochondrial membrane potential. Intriguingly, the characteristics of typical apoptosis such as phosphatidylserine externalization and DNA fragmentation were not detected in the cell death process caused by this PDT regime. In conclusion, our results show that when plasma membranes are the main targets, Photofrin-PDT can lead to instant ROS formation and subsequent activation of downstream signaling events similar to those elicited by many apoptotic stimuli, but the damage of plasma membranes renders the death phenotype more necrosis like.     

A Perspective on Neuronal Cell Death Signaling and Neurodegeneration

Although neuronal cell death through apoptotic pathways represents a common feature of dysferopathies, the canonical apoptotic changes familiar from nonneuronal cells are late events. Loss of neuronal function occurs at a much early time, when synaptic-based neuronal connectivity fails. In this context, apoptotic pathways may normally serve a cleanup role, rather than a pathogenic one. Reframing the consideration of cell death in the nervous system to include the early stages of axonal degeneration provides a better understanding of the roles played by various apoptotic signaling pathways in neurodegenerative diseases. Focusing on disease-specific mechanisms that initiate the sequence that eventually leads to neuronal loss should facilitate development of therapies that preserve neuronal function and neuronal numbers.     

Anti-apoptotic effects of curcumin on photosensitized human epidermal carcinoma A431 cells

Photodynamic treatment (PDT) can elicit a diverse range of cellular responses, including apoptotic cell death. Previously, we showed that PDT stimulates caspase-3 activation and subsequent cleavage and activation of p21-activated kinase 2 (PAK2) in human epidermal carcinoma A431 cells. Curcumin, the yellow pigment of Curcuma longa, is known to have anti-oxidant and anti-inflammatory properties. In the present study, using Rose Bengal (RB) as the photosensitizer, we investigated the effect of curcumin on PDT-induced apoptotic events in human epidermal carcinoma A431 cells. We report that curcumin prevented PDT-induced JNK activation, mitochondrial release of cytochrome c, caspase-3 activation, and cleavage of PAK2. Using the cell permeable dye DCF-DA as an indicator of reactive oxygen species (ROS) generation, we found that both curcumin and ROS scavengers (i.e., l-histidine, a-tocopherol, mannitol) abolished PDT-stimulated intracellular oxidative stress. Moreover, all these PDT-induced apoptotic changes in cells could be blocked by singlet oxygen scavengers (i.e., l-histidine, a-tocopherol), but were not affected by the hydroxyl radical scavenger mannitol. In addition, we found that SP600125, a JNK-specific inhibitor, reduced PDT-induced JNK activation as well as caspase-3 activation, indicating that JNK activity is required for PDT-induced caspase activation. Collectively, these results demonstrate that singlet oxygen triggers JNK activation, cytochrome c release, caspase activation and subsequent apoptotic biochemical changes during PDT and show that curcumin is a potent inhibitor for this process.     

The morphology of apoptosis

The concept of apoptotic cell death as an essential part of the development and life of complex organisms has been devised in different situations and tested from various angles. This review article discusses the morphological changes during death by apoptosis. In cells undergoing apoptosis, an intracellular signalling pathway operates cell autonomously to implement the death and disposal of the cell. The similarity of the biochemical events during apoptosis in different situations is reflected by a high uniformity of morphological changes in many situations of naturally occurring or experimentally induced cell death. The unifying concept of apoptosis has been derived from the observation of this morphological consistency of dying cells almost 30 years ago. Since then, we have learned much about the intracellular signalling in the apoptotic process and the molecular background has been delineated which guides the initiation of the morphological changes. Here, an attempt is made to present the current knowledge about the molecular events in the development of these morphological alterations and to place these changes in the context of apoptotic cell death.     

Modality of Cell Death Induced by Foscan®-Based Photodynamic Treatment in Human Colon Adenocarcinoma Cell Line HT29

Apoptosis induced by photodynamic therapy (PDT) is considered to be an important factor defining the treatment outcome. Nevertheless, the relevance of apoptotic events in overall cell death should be established for every given photosensitizer. The present study addresses the contribution of Foscan-(meta-tetra(hydroxyphenyl)chlorine; mTHPC) photosensitized apoptosis in overall cell death in a model of cultured HT29 adenocarcinoma cells. Early events of cell death were assessed by the evaluation of mitochondrial response to mTHPC-mediated PDT, cytochrome c release and membrane depolarization. Apoptosis was measured through the activity of caspase-3 and the binding of the fluorescent conjugate Ca2+-dependent protein Annexin-V on membrane externalized phosphatidylserine at 2, 4, and 24 h post-PDT. Immediately after mTHPC-PDT, from 28 to 57% cells exhibited cytochrome c release concomitantly with mitochondrial membrane depolarization for light doses inducing more than 90% overall cell death. The maximum of caspase-3 activation (12-fold more than control) was reached 24 h after irradiation at fluence inducing 90% cell death (LD(90)). The corresponding measurement of apoptotic cells (12% of Annexin-V bound cells) confirmed the mild and delayed apoptotic response of HT29 cells to mTHPC-PDT.     

Regulatory pathways in photodynamic therapy induced apoptosis.

Photodynamic therapy is an approved treatment for several types of tumors and certain benign diseases, based on the use of a light-absorbing compound (photosensitizer) and light irradiation. In the presence of molecular oxygen, light-activation of the photosensitizer, which accumulates in cancer tissues, leads to the local production of reactive oxygen species that kill the tumor cells. Mitochondria are central coordinators of the mechanisms by which PDT induces apoptosis in the target cells. Recent studies indicate that concomitant to the permeabilization of the outer mitochondrial membrane (which leads to the release of several apoptogenic factors in the cytosol and to the activation of effector caspases), regulatory signaling pathways are activated in a photosensitizer, PDT dose and cell-dependent fashion. Signaling pathways regulated by members of mitogen activated protein kinases and their downstream targets, such as cyclooxygenase-2, appear to critically modulate cancer cell sensitivity to PDT. Understanding the molecular events that contribute to PDT-induced apoptosis, and how cancer cells can evade apoptotic death, should enable a more rationale approach to drug design and therapy.     

Photodynamic therapy: shedding light on the biochemical pathways regulating porphyrin-mediated cell death

Photodynamic therapy (PDT) is a clinically approved treatment for the ocular condition age-related macular degeneration, and certain types of cancer. PDT is also under investigation for other ocular, as well as, immune-mediated and cardiovascular indications. PDT is a two step procedure. In the first step, the photosensitizer, usually a porphyrin derivative, is administered and taken up by cells. The second step involves activation of the photosensitizer with a specific wavelength of visible light. Exposure to light of an activating wavelength generates reactive oxygen species within cells containing photosensitizer. PDT with porphyrin photosensitizers induces rapid apoptotic cell death, an event which may be attributed to the close association of these compounds with mitochondria. Thus, PDT is an attractive method to treat ailments such as cancer, viral infections, autoimmune disorders and certain cardiovascular diseases in which the apoptotic program may be compromised. The present review examines the cellular events triggered at lethal and sublethal PDT doses and their relationship to the subsequent effects exerted upon cells.     

光动力疗法中细胞凋亡的几种信号转导路径

光动力疗法是一种新型的治疗肿瘤的方法。光敏剂在肿瘤细胞中积累,受光激发后,产生毒性的活性氧,杀死肿瘤细胞。在光动力疗法中,细胞凋亡是细胞死亡的一种重要的形式。本文主要总结了死亡受体、线粒体和介导的凋亡信号转导路径。     

Photodynamic activity of a new sensitizer derived from porphyrin-C60 dyad and its biological consequences in a human carcinoma cell line

The photokilling activity of a porphyrin-C₆₀ (P-C₆₀) dyad was evaluated on a Hep-2 human larynx-carcinoma cell line. This study represents the first evaluation of a dyad, with high capacity to form a photoinduced charge-separated state, to act as agent to inactivate cells by photodynamic therapy (PDT). Cell treatment was carried out with 1 μM P-C₆₀ incorporated into liposomal vesicles. No dark cytotoxicity was observed using 1 μM P-C₆₀ concentration and during long incubation time (24 h). The uptake of sensitizer into Hep-2 was studied at different times of incubation. Under these conditions, a value of 1.5 nmol/10⁶ cells was found after 4 h of incubation showing practically no change even after 24 h. The cell survival after irradiation of the cells with visible light was dependent upon light exposure level. A high photocytotoxic effect was observed for P-C₆₀, which inactivated 80% of the cells after 54 J/cm² of irradiation. Moreover, the dyad kept a high photoactivity even under argon atmosphere. Thus, depending on the microenviroment where the sensitizer is localized, this compound could produce a biological photodamage through either a ¹O₂-mediated photoreaction process or a free radical mechanism under low oxygen concentration.

The mechanism of cell death was analyzed by Hoechst-33258, toluidine blue staining, TUNEL and DNA fragmentation. Cell cultures treated for 24 h with P-C₆₀ and irradiated with a dose of 54 J/cm² showed a great amount of apoptotic cells (58%). Moreover, changes in cell morphology were analyzed using fluorescence microscopy with Hoechst-33258 under low oxygen concentration. Under this anaerobic condition, necrotic cellular death predominated on apoptotic pathway. There were more apoptotic cells under air irradiation condition than under argon irradiation condition. To determine the apoptotic pathway, caspase-3 activation was studied by caspase-3 activity detection kits. The last results showed that P-C₆₀ induced apoptosis by caspase-3-dependent pathway. These results indicated that molecular dyad, which can form a photoinduced charge-separated state, is a promising model for phototherapeutic agents and they have potential application in cell inactivation by PDT.     

Molecular effectors of multiple cell death pathways initiated by photodynamic therapy

Photodynamic therapy (PDT) is a recently developed anticancer modality utilizing the generation of singlet oxygen and other reactive oxygen species, through visible light irradiation of a photosensitive dye accumulated in the cancerous tissue. Multiple signaling cascades are concomitantly activated in cancer cells exposed to the photodynamic stress and depending on the subcellular localization of the damaging ROS, these signals are transduced into adaptive or cell death responses. Recent evidence indicates that PDT can kill cancer cells directly by the efficient induction of apoptotic as well as non-apoptotic cell death pathways. The identification of the molecular effectors regulating the cross-talk between apoptosis and other major cell death subroutines (e.g. necrosis, autophagic cell death) is an area of intense research in cancer therapy. Signaling molecules modulating the induction of different cell death pathways can become useful targets to induce or increase photokilling in cancer cells harboring defects in apoptotic pathways, which is a crucial step in carcinogenesis and therapy resistance. This review highlights recent developments aimed at deciphering the molecular interplay between cell death pathways as well as their possible therapeutic exploitation in photosensitized cells.     

Cell death via mitochondrial apoptotic pathway due to activation of Bax by lysosomal photodamage

Lysosomal photosensitizers have been used in photodynamic therapy. The combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. Lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, N-aspartyl chlorin e6 (NPe6), an effective photosensitizer that preferentially accumulates in lysosomes, was used to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells (ASTC-a-1) after NPe6-photodynamic treatment (NPe6-PDT) was studied using real-time single-cell analysis. Our results demonstrated that NPe6-PDT induced rapid generation of reactive oxygen species (ROS). The photodynamically produced ROS caused a rapid destruction of lysosomes, leading to release of cathepsins, and the ROS scavengers vitamin C and NAC prevent the effects. Then the following spatiotemporal sequence of cellular events was observed during cell apoptosis: Bcl-2-associated X protein (Bax) activation, cytochrome c release, and caspase-9/-3 activation. Importantly, the activation of Bax proved to be a crucial event in this apoptotic machinery, because suppressing the endogenous Bax using siRNA could significantly inhibit cytochrome c release and caspase-9/-3 activation and protect the cell from death. In conclusion, this study demonstrates that PDT with lysosomal photosensitizer induces Bax activation and subsequently initiates the mitochondrial apoptotic pathway.     

Evaluation of photodynamic treatment using aluminum phthalocyanine tetrasulfonate chloride as a photosensitizer: new approach

Photodynamic therapy (PDT) has been the subject of several clinical studies. Evidence to date suggests that direct cell death may involve apoptosis. T(24) cells (bladder cancer cells, ATCC-Nr. HTB-4) were subjected to PDT with aluminum phthalocyanine tetrasulfonate chloride (AlS(4)Pc-Cl) and red laser light at 670 nm. Morphological changes after PDT were visualized under confocal microscopy. Raman microspectroscopy is considered as one of the newly established methods used for the detection of cytochrome c as an apoptotic marker. Results showed that PDT treated T(24) cells seem to undergo apoptosis after irradiation with 3 J cm(-2). Cytochrome c could not be detected from cells incubated with AlS(4)Pc-Cl using Raman spectroscopy whereas AlS(4)Pc-Cl seems to interfere with the Raman spectrum of cytochrome c.     

Mechanism of cell death by 5‐aminolevulinic acid‐based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937

Photodynamic therapy (PDT) for tumors is based on the tumor‐selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5‐aminolevulinic acid (ALA)‐based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl‐xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase‐3 activation, phosphatidylserine (PS) externalization. PDT‐induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA‐based‐PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA. Copyright © 2009 John Wiley & Sons, Ltd.     

The small GTPase Cdc42 initiates an apoptotic signaling pathway in Jurkat T lymphocytes.

Apoptosis plays an important role in regulating development and homeostasis of the immune system, yet the elements of the signaling pathways that control cell death have not been well defined. When expressed in Jurkat T cells, an activated form of the small GTPase Cdc42 induces cell death exhibiting the characteristics of apoptosis. The death response induced by Cdc42 is mediated by activation of a protein kinase cascade leading to stimulation of c-Jun amino terminal kinase (JNK). Apoptosis initiated by Cdc42 is inhibited by dominant negative components of the JNK cascade and by reagents that block activity of the ICE protease (caspase) family, suggesting that stimulation of the JNK kinase cascade can lead to caspase activation. The sequence of morphological events observed typically in apoptotic cells is modified in the presence of activated Cdc42, suggesting that this GTPase may account for some aspects of cytoskeletal regulation during the apoptotic program. These data suggest a means through which the biochemical and morphological events occurring during apoptosis may be coordinately regulated.     

Apoptotic cell death following traumatic injury to the central nervous system

Apoptotic cell death is a fundamental and highly regulated biological process in which a cell is instructed to actively participate in its own demise. This process of cellular suicide is activated by developmental and environmental cues and normally plays an essential role in eliminating superfluous, damaged, and senescent cells of many tissue types. In recent years, a number of experimental studies have provided evidence of widespread neuronal and glial apoptosis following injury to the central nervous system (CNS). These studies indicate that injury-induced apoptosis can be detected from hours to days following injury and may contribute to neurological dysfunction. Given these findings, understanding the biochemical signaling events controlling apoptosis is a first step towards developing therapeutic agents that target this cell death process. This review will focus on molecular cell death pathways that are responsible for generating the apoptotic phenotype. It will also summarize what is currently known about the apoptotic signals that are activated in the injured CNS, and what potential strategies might be pursued to reduce this cell death process as a means to promote functional recovery.     

Fas Induces Cytoplasmic Apoptotic Responses and Activation of the MKK7-JNK/SAPK and MKK6-p38 Pathways Independent of CPP32-like Proteases

IL-1β converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-, CPP32-, and Ich-1–like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases which is thought to be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. Here, we have shown that MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and that MKK6 is a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors did not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by the dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.     

Vertebrate cell death in energy-limited conditions and how to avoid it: what we might learn from mammalian hibernators and other stress-tolerant vertebrates

Dormancy in vertebrates may expose cells to acidosis, hypoxia/anoxia, oxidative damage, and extremes in temperature. All of these insults are known to be pro-apoptotic in typical vertebrate cells, especially mammals. Since dormancy is presumably the result of a need for energy conservation, the inherent energetic demand of replenishing cells that underwent apoptosis seems at odds with this strategy. This review will discuss processes to mitigate apoptosis and how these processes might be regulated in stress-tolerant vertebrates such as mammalian hibernators. As data directly addressing such issues are scarce and often conflicting, an apparently complex regulation of apoptosis seems to be at work. For example, apoptosis is mitigated during dormancy, key signaling events including the activation of caspase-3 may still occur. However, both passive, temperature-induced depression of apoptotic signaling as well as active suppression of apoptosis appear to work in synergy in these systems. In many instances cell death is prevented by simply avoiding the cellular triggers (e.g. leakage of proteins from the mitochondria or increases in intracellular calcium) that initiate apoptotic signaling. In this review we discuss what is known about programmed cell death in these under-studied models and highlight features of their physiology that likely support survival in the face of conditions that would induce cell death in typical vertebrate cells.     

Resistance against ricin-induced apoptosis in a brefeldin A-resistant mutant cell line (BER-40) of Vero cells

We have found that a brefeldin A (BFA)-resistant mutant cell line derived from Vero cells (BER-40) is highly resistant to ricin-induced apoptosis as compared with parental Vero cells. In BER-40 cells, all apoptotic events caused by ricin including cytolysis, nuclear morphological changes, and DNA fragmentation occur to a lesser extent than in Vero cells, even though both cell lines show similar sensitivities to ricin-mediated inhibition of protein synthesis. Furthermore, no significant apoptotic signaling events, such as increases in caspase-3 and -9-like activities, release of cytochrome c from mitochondria, or the cleavage of PARP, were observed in BER-40 cells under the conditions at which these changes were evident in Vero cells. Intracellular biochemical changes associated with ricin-induced apoptosis, such as the depletion of glutathione and an increase in free Zn2+, were also less apparent in BER-40 cells than in Vero cells. BER-40 cells were also found to be highly resistant to apoptosis induced by other toxins with different intoxication mechanisms such as diphtheria toxin, modeccin, and anisomycin. These results suggest that the entire apoptotic signal transduction mechanism in BER-40 cells, which may be triggered after the inhibition of protein synthesis by toxins, becomes resistant. Since MDCK cells, a naturally BFA resistant cell line, are highly sensitive to ricin-induced apoptosis, it seems likely that the BFA resistance phenotype may not necessarily lead to resistance to apoptotic cell death. Probably the underlaying BFA-resistance mechanism in BER-40 cells is distinct from that in MDCK cells, and the resistance to ricin-induced apoptosis of BER-40 cells may be a unique phenotype acquired concomitantly with BFA-resistance.