Apoptotic and autophagic responses to Bcl-2 inhibition and photodamage (original) (raw)
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Initiation of apoptosis and autophagy by photodynamic therapy
Lasers in surgery and medicine, 2006
This study was designed to examine modes of cell death after photodynamic therapy (PDT). Murine leukemia L1210 cells and human prostate Bax-deficient DU145 cells were examined after PDT-induced photodamage to the endoplasmic reticulum (ER). Phase contrast, fluorescence and electron microscopy were used to identify changes in cellular morphology, chromatin condensation, loss of mitochondrial membrane potential, and formation of phagolysosomes. Western blots were used to assess the processing of LC3-I to LC3-II, a marker for autophagy. Inhibitors of apoptosis and/or autophagy were used to delineate the contributions of the two pathways to the effects of PDT. Both apoptosis and autophagy occurred in L1210 after ER photodamage with the latter predominating after 24 hours. In DU145 cells, PDT conditions causing comparable cytotoxicity only initiated autophagy. PI3-kinase inhibitors suppressed autophagy in both cell lines as indicated by inhibition of vacuolization and LC3 processing. Bot...
Apoptosis and Autophagy After Mitochondrial or Endoplasmic Reticulum Photodamage
Photochemistry and Photobiology, 2007
Photodynamic therapy (PDT) can cause lethal photodamage by both direct and indirect mechanisms. Direct modes of cell death relate to nonspecific necrosis and the initiation of signaling pathways that elicit apoptosis, autophagy or both. In this report, effects of low-dose and high-dose PDT are explored, comparing sensitizers that localize in the endoplasmic reticulum (the porphycene termed CPO) or mitochondria (mesochlorin). To explore the role of autophagy, two cell lines were examined -the murine L1210 leukemia and an Atg7 knockdown derivative of L1210. The Atg7 gene is central to the process of autophagy. High-dose PDT with either sensitizer resulted in a substantial loss of the Bcl-2 protein. As Bcl-2 regulates both apoptosis and autophagy, loss of this protein can lead to initiation of either or both processes. Low-dose PDT with either sensitizer resulted in the initiation of apoptosis in the L1210/Atg7 − cell line and a 20% loss of viability. In contrast, the same PDT dose led to the rapid appearance of autophagic cells in the L1210 line, less apoptosis and only a 5% loss of viability. These results are consistent with autophagy serving as a pro-survival response via the recycling of damaged organelles. At a higher PDT dose more apoptosis was again seen in the L1210/ Atg7 − line, but both cell lines exhibited comparable cytotoxicity in colony formation assays. We conclude that autophagy offers protection from the phototoxic effects of low-dose PDT, but can serve as an alternate death mode when the PDT dose is increased. † This invited paper is part of the Symposium-in-Print: Photodynamic Therapy.
Photodynamic Therapy and Cell Death Pathways
Methods in Molecular Biology, 2010
Photodynamic therapy (PDT) is the term used to describe the irradiation of photosensitized cells or tissue with phototoxic consequences. This process can result in the rapid initiation of not only apoptosis, an irreversible death pathway, but also autophagy. The procedures described here are designed to characterize the correlation between the PDT dose vs. survival of cells in vitro, the apoptotic effects of photodamage, and the extent of an autophagic response. These are assessed by clonogenic assays, observation of condensed chromatin characteristic of apoptosis, activation of "executioner" caspases, and the autophagic flux as indicated by comparing accumulation of the LC3-II protein under conditions where processing of autophagosomes is retarded vs. is not retarded.
Death pathways associated with photodynamic therapy
Medical laser application : international journal for laser treatment and research, 2006
When the mitochondria and/or the endoplasmic reticulum were targeted by photodynamic therapy, photodamage to the anti-apoptotic protein Bcl-2 was observed. This led to an apoptotic outcome if that death pathway was available. Lysosomal photodamage ultimately resulted in activation of the pro-apoptotic protein Bid, also leading to apoptosis. Photodamage to the plasma membrane was associated with migration of sensitizers to the cytosol and procaspase photodamage, with apoptosis impaired. Where apoptosis was unavailable because of lack of necessary components of the program, an autophagic outcome has been observed. It is also clear that autophagy can occur along with apoptosis as a PDT response, and may play a role in immunologic responses to photodamaged tumor cells.
Assessing autophagy in the context of photodynamic therapy
Autophagy, 2010
Photodynamic therapy (PDT) is a procedure that has applications in the selective eradication of neoplasia where sites of malignant lesions are clearly delineated. It is a two-step process whereby cells are first sensitized to light and then photoirradiated. This results in the formation of singlet molecular oxygen and other reactive oxygen species that can cause photodamage at sites where the photosensitizing agent has localized. Photosensitizers found to be clinically useful show affinity for the endoplasmic reticulum (ER), mitochondria, lysosomes, or combinations of these sites. The induction of apoptosis and/or autophagy in photosensitized cells is a common outcome of PDT. This report explores the following issues: (1) Does the induction of autophagy in PDT protocols occur independent of, or in association with, apoptosis? (2) Does the resulting autophagy play a prosurvival or prodeath role? (3) Do photosensitizers damage/inactivate specific proteins that are components of, or that modulate the autophagic process? (4) Can an autophagic response be mounted in cells in which lysosomes are specifically photodamaged? In brief, autophagy can occur independently of apoptosis in PDT protocols, and appears to play a prosurvival role in apoptosis competent cells, and a prodeath role in apoptosis incompetent cells. Mitochondrial and ER-localized sensitizers cause selective photodamage to some (i.e., Bcl-2, Bcl-x L , mTOR) proteins involved in the apoptotic/ autophagic process. Finally, an aborted autophagic response occurs in cells with photodamaged lysosomes. Whereas autophagosomes form, digestion of their cargo is compromised because of the absence of functional lysosomes.
Chapter 1 Initiation of Autophagy by Photodynamic Therapy
Methods in Enzymology, 2009
Photodynamic therapy (PDT) involves the irradiation of photosensitized cells with light. Depending on localization of the photosensitizing agent, the process can induce photodamage to the endoplasmic reticulum (ER), mitochondria, plasma membrane, and/or lysosomes. When ER or mitochondria are targeted, antiapoptotic proteins of the Bcl-2 family are especially sensitive to photodamage. Both apoptosis and autophagy can occur after PDT, autophagy being associated with enhanced survival at low levels of photodamage to some cells. Autophagy can become a cell-death pathway if apoptosis is inhibited or when cells attempt to recycle damaged constituents beyond their capacity for recovery. While techniques associated with characterization of autophagy are generally applicable, PDT introduces additional factors related to unknown sites of photodamage that may alter autophagic pathways. This chapter discusses issues that may arise in assessing autophagy after cellular photodamage. the conversion of molecular oxygen to a reactive oxygen species (ROS) termed singlet oxygen.
Subcellular targets for photodynamic therapy: implications for initiation of apoptosis and autophagy
Journal of the National Comprehensive Cancer Network : JNCCN, 2012
Direct lethal effects of photodynamic therapy (PDT) on a cell population were initially shown to occur via initiation of apoptosis, with high doses having a necrotic effect. Selective induction of cellular "self-digestion," better known as autophagy, represents a novel therapeutic target for the prevention of tumor growth and metastasis. It should be noted that autophagy has conflicting roles in the regulation of cell death, which, when applied to oncology, may produce both positive and negative effects on tumor development. Through better understanding the complex parts played by autophagy among diverse cellular signaling pathways in preclinical models, it may be possible to selectively regulate autophagy in response to specific stimuli, thereby inhibiting its oncogenic tendencies while preserving its tumor-suppressive capabilities.
Cell Death Pathways in Photodynamic Therapy of Cancer
Cancers, 2011
Photodynamic therapy (PDT) is an emerging cancer therapy that uses the combination of non-toxic dyes or photosensitizers (PS) and harmless visible light to produce reactive oxygen species and destroy tumors. The PS can be localized in various organelles such as mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus and plasma membranes and this sub-cellular location governs much of the signaling that occurs after PDT. There is an acute stress response that leads to changes in calcium and lipid metabolism and causes the production of cytokines and stress response mediators. Enzymes (particularly protein kinases) are activated and transcription factors are expressed. Many of the cellular responses center on mitochondria and frequently lead to induction of apoptosis by the mitochondrial pathway involving caspase activation and release of cytochrome c. Certain specific proteins (such as Bcl-2) are damaged by PDT-induced oxidation thereby increasing apoptosis, and a build-up of oxidized proteins leads to an ER-stress response that may be increased by proteasome inhibition. Autophagy plays a role in either inhibiting or enhancing cell death after PDT.
Cell Calcium, 2001
Photodynamic therapy (PDT) is clinically approved for the treatment of several types of cancer as well as agerelated macular degeneration, the leading cause of blindness in the elderly. PDT using the photosensitizer verteporfin has been previously shown to induce rapid apoptosis via a mitochondrial-caspase activation pathway. The impact of PDT on other cellular organelles such as the endoplasmic reticulum (ER) is undefined. The effect of PDT on intracellular Ca 2; ([Ca 2; ] i ) in control and Bcl-2-overexpressing HeLa cells was assessed. A greater [Ca 2; ] i transient was observed for Bcl-2 overexpressing cells in response to PDT. The PDT-induced Ca 2; release was due to the emptying of Ca 2; from ER and possibly mitochondrial stores and was not due to an influx of Ca 2; from the medium. For Bcl-2-transfected cells, the release of Ca 2; was incomplete as determined by a further [Ca 2; ] i transient produced by the addition of the Ca 2; ionophore ionomycin after PDT. Furthermore, extrusion of Ca 2; was not hindered while ER-mediated sequestration of Ca 2; was impaired after PDT. Impairment of ER-mediated sequestration of Ca 2; may be due to the immediate caspase-independent depletion of sarco/endoplasmic reticulum Ca 2; ATPase-2 (SERCA2) that occurred in response to PDT in birth HeLa/Neo and Bcl-2 overexpressed HeLa cells. In summary, PDT induced the rapid degradation of SERCA2 and release of ER and mitochondrial Ca 2; stores. Although overexpression of Bcl-2 did not protect against SERCA2 degradation, it may influence the release of Ca 2; from ER and mitochondrial stores in PDT-treated cells.