Hypericin‐PDT‐induced rapid necrotic death in human squamous cell carcinoma cultures after multiple treatment

PDT (photodynamic therapy) has been used for the treatment of NMCC (non‐melanoma cutaneous cancer) particularly, human SCC (squamous cell carcinoma). However, the nature of the photosensitizer, the activation light source and the mode of cell death induced post‐PDT remains elusive. We tried to optimize PDT using the light‐activated (320–400 nm) St John's Wort‐derived compound, Hyp (hypericin). The work highlights the potential mode of cell death and the increased efficacy of the technique associated with multiple Hyp‐PDT treatment. SCC cells were exposed to different concentrations of Hyp and activated with light at 1 J/cm² for 1 or 2 days. Thereafter with the optimum dose of Hyp proliferation, ROS (reactive oxygen species), and apoptosis were analysed by XTT [2,3‐bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide] assay, FACS analysis and Fluorescent/Phase contrast microscopy was carried out for morphological studies. Hyp‐PDT produces more ROS after 1 day compared with 2 days and the mode of cell death is a necrotic caspase‐independent mechanism. We propose a novel ‘double‐hit/2‐day’ strategy to reduce the viability in SCC using Hyp‐based PDT as an adjunctive treatment modality.     

High fluence low‐power laser irradiation induces mitochondrial permeability transition mediated by reactive oxygen species

High fluence low‐power laser irradiation (HF‐LPLI) can induce cell apoptosis via the mitochondria/caspase‐3 pathway. Here, we further investigated the mechanism involved in the apoptotic process in human lung adenocarcinoma cells (ASTC‐a‐1) at a laser irradiation fluence of 120 J/cm² (633 nm). Cytochrome c release was ascribed to mitochondrial permeability transition (MPT) because the release was prevented by cyclosporine (CsA), a specific inhibitor of MPT. Furthermore, mitochondrial permeability for calcein (～620 Da) was another evidence for the MPT induction under HF‐LPLI treatment. A high‐level intracellular reactive oxygen species (ROS) generation was observed after irradiation. The photodynamically produced ROS caused onset of MPT, as the ROS scavenger docosahexaenoic acid (DHA) prevented the MPT. However, CsA failed to prevented cell death induced by HF‐LPLI, indicating the existence of other signaling pathways. Following laser irradiation, Bax activation occurred after mitochondrial depolarization and cytochrome c release, indicating Bax activation was a downstream event. In the presence of CsA, Bax was still activated at the end‐stage of apoptotic process caused by HF‐LPLI, suggesting that Bax was involved in an alternative‐signaling pathway, which was independent of MPT. Under HF‐LPLI treatment, cell viabilities due to pre‐treatment with DHA, CsA, or Bax small interfering RNA (siRNA) demonstrated that the MPT signaling pathway was dominant, while Bax signaling pathway was secondary, and more importantly ROS mediated both pathways. Taken together, these results showed that HF‐LPLI induced cell apoptosis via the CsA‐sensitive MPT, which was ROS‐dependent. Furthermore, there existed a secondary signaling pathway through Bax activation. The observed link between MPT and triggering ROS could be a fundamental phenomenon in HF‐LPLI‐induced cell apoptosis. J. Cell. Physiol. 218: 603–611, 2009. © 2008 Wiley‐Liss, Inc.     

LED Light-Induced ROS Differentially Regulates Focal Adhesion Kinase Activity in HaCaT Cell Viability

In this study, changes in cell signaling mechanisms in skin cells induced by various wavelengths and intensities of light-emitting diodes (LED) were investigated, focusing on the activity of focal adhesion kinase (FAK) in particular. We examined the effect of LED irradiation on cell survival, the generation of intracellular reactive oxygen species (ROS), and the activity of various cell-signaling proteins. Red LED light increased cell viability at all intensities, whereas strong green and blue LED light reduced cell viability, and this effect was reversed by NAC or DPI treatment. Red LED light caused an increase in ROS formation according to the increase in the intensity of the LED light, and green and blue LED lights led to sharp increases in ROS formation. In the initial reaction to LEDs, red LED light only increased the phosphorylation of FAK and extracellular-signal regulated protein kinase (ERK), whereas green and blue LED lights increased the phosphorylation of inhibitory-κB Kinase α (IKKα), c-jun N-terminal kinase (JNK), and p38. The phosphorylation of these intracellular proteins was reduced via FAK inhibitor, NAC, and DPI treatments. Even after 24 h of LED irradiation, the activity of FAK and ERK appeared in cells treated with red LED light but did not appear in cells treated with green and blue LED lights. Furthermore, the activity of caspase-3 was confirmed along with cell detachment. Therefore, our results suggest that red LED light induced mitogenic effects via low levels of ROS–FAK–ERK, while green and blue LED lights induced cytotoxic effects via cellular stress and apoptosis signaling resulting from high levels of ROS.     

Differential cytotoxic responses to low- and high-dose photodynamic therapy in human gastric and bladder cancer cells

Here, we present differential cytotoxic responses to two different doses of photodynamic therapies (PDTs; low-dose PDT [LDP] and high-dose PDT [HDP]) using a chlorin-based photosensitizer, DH-II-24, in human gastric and bladder cancer cells. Fluorescence-activated cell sorting analysis using Annexin V and propidium iodide (PI) showed that LDP induced apoptotic cell death, whereas HDP predominantly caused necrotic cell death. The differential cytotoxic responses to the two PDTs were further confirmed by a DiOC(6) and PI double-staining assay via confocal microscopy. LDP, but not HDP, activated caspase-3, which was inhibited by Z-VAD, Trolox, and BAPTA-AM. LDP and HDP demonstrated opposite effects on intracellular reactive oxygen species (ROS)/Ca(2+) signals; LDP stimulated intracellular ROS production, contributing to a transient increase of intracellular Ca(2+) , whereas HDP induced a massive and prolonged elevation of intracellular Ca(2+) responsible for the transient production of intracellular ROS. In addition, the two PDTs also increased in situ transglutaminase 2 (TG2) activity, with a higher stimulation by HDP, and this increase in activity was prevented by Trolox, BAPTA-AM, and TG2-siRNA. LDP-induced apoptotic cell death was strongly inhibited by Trolox and TG2-siRNA and moderately suppressed by BAPTA-AM. However, HDP-mediated necrotic cell death was partially inhibited by BAPTA-AM but not by TG2-siRNA. Thus, these results demonstrate that LDP and HDP induced apoptotic and necrotic cell death by differential signaling mechanisms involving intracellular Ca(2+) , ROS, and TG2.     

Blue light emitting diode induces apoptosis in lymphoid cells by stimulating autophagy

The present study was performed to examine the induction of apoptotic cell death and autophagy by blue LED irradiation, and the contribution of autophagy to apoptosis in B cell lymphoma A20 and RAMOS cells exposed to blue LED. Irradiation with blue LED reduced cell viability and induced apoptotic cell death, as indicated by exposure of phosphatidylserine on the plasma outside membrane and fragmentation of DNA. Furthermore, the mitochondrial membrane potential increased, and apoptotic proteins (PARP, caspase 3, Bax, and bcl-2) were observed. In addition, the level of intracellular superoxide anion (O₂⁻) gradually increased. Interestingly the formation of autophagosomes and level of LC3-II were increased in blue LED-irradiated A20 and RAMOS cells, but inhibited after pretreatment with 3-methyladenine (3-MA), widely used as an autophagy inhibitor. Inhibition of the autophagic process by pretreatment with 3-MA blocked blue LED irradiation-induced caspase-3 activation. Moreover, a significant reduction of both the early and late phases of apoptosis after transfection with ATG5 and beclin 1 siRNAs was shown by the annexin V/PI staining, indicating a crucial role of autophagy in blue LED-induced apoptosis in cells. Additionally, the survival rate of mice irradiated with blue LED after injection with A20 cells increased compared to the control group. Our data demonstrate that blue LED irradiation induces apoptosis via the mitochondrial-mediated pathway, in conjunction with autophagy. Further studies are needed to elucidate the precise mechanism of blue LED-induced immune cell death.     

Blue LED causes autophagic cell death in human osteosarcoma by increasing ROS generation and dephosphorylating EGFR

Osteosarcoma (OS) is the most common primary malignant bone tumour in adolescence. Lately, light-emitting diodes (LED)-based therapy has emerged as a new promising approach for several diseases. However, it remains unknown in human OS. Here, we found that the blue LED irradiation significantly suppressed the proliferation, migration and invasion of human OS cells, while we observed blue LED irradiation increased ROS production through increased NADPH oxidase enzymes NOX2 and NOX4, as well as decreased Catalase (CAT) expression levels. Furthermore, we revealed blue LED irradiation-induced autophagy characterized by alterations in autophagy protein markers including Beclin-1, LC3-II/LC3-I and P62. Moreover, we demonstrated an enhanced autophagic flux. The blockage of autophagy displayed a remarkable attenuation of anti-tumour activities of blue LED irradiation. Next, ROS scavenger N-acetyl-L-cysteine (NAC) and NOX inhibitor diphenyleneiodonium (DPI) blocked suppression of OS cell growth, indicating that ROS accumulation might play an essential role in blue LED-induced autophagic OS cell death. Additionally, we observed blue LED irradiation decreased EGFR activation (phosphorylation), which in turn led to Beclin-1 release and subsequent autophagy activation in OS cells. Analysis of EGFR colocalization with Beclin-1 and EGFR-immunoprecipitation (IP) assay further revealed the decreased interaction of EGFR and Beclin-1 upon blue LED irradiation in OS cells. In addition, Beclin-1 down-regulation abolished the effects of blue LED irradiation on OS cells. Collectively, we concluded that blue LED irradiation exhibited anti-tumour effects on OS by triggering ROS and EGFR/Beclin-1-mediated autophagy signalling pathway, representing a potential approach for human OS treatment.     

Cellular transformations in near‐infrared light‐induced apoptosis in cancer cells revealed by label‐free CARS imaging

Photobiomodulation (PBM) involves light to activate cellular signaling pathways leading to cell proliferation or death. In this work, fluorescence and Coherent anti‐Stokes Raman Scattering (CARS) imaging techniques were applied to assess apoptosis in human cervical cancer cells (HeLa) induced by near infrared (NIR) laser light (808 nm). Using the Caspase 3/7 fluorescent probe to identify apoptotic cells, we found that the pro‐apoptotic effect is significantly dependent of irradiation dose. The highest apoptosis rate was noted for the lower irradiation doses, that is, 0.3 J/cm² (~58%) and 3 J/cm² (~28%). The impact of light doses on proteins/lipids intracellular metabolism and distribution was evaluated using CARS imaging, which revealed apoptosis‐associated reorganization of nuclear proteins and cytoplasmic lipids after irradiation with 0.3 J/cm². Doses of NIR light causing apoptosis (0.3, 3 and 30 J/cm²) induced a gradual increase in the nuclear protein level over time, in contrast to proteins in cells non‐irradiated and irradiated with 10 J/cm². Furthermore, irradiation of the cells with the 0.3 J/cm² dose resulted in lipid droplets (LDs) accumulation, which was apparently caused by an increase in reactive oxygen species (ROS) generation. We suggest that PBM induced apoptosis could be caused by the ability of NIR light to trigger excessive LDs formation which, in turn, induces cellular cytotoxicity.      

Effect of blue light‐emitting diode light and antioxidant potential in a somatic cell

Light is an indispensable part of routine laboratory work in which conventional light is generally used. Light‐emitting diodes (LEDs) have come to replace conventional light, and thus could be a potent target in biomedical studies. Since blue light is a major component of visible light wavelength, in this study, using a somatic cell from the African green monkey kidney, we assessed the possible consequences of the blue spectra of LED light in future animal experiments and proposed a potent mitigation against light‐induced damage. COS‐7 cells were exposed to blue LED light (450 nm) and the growth and deoxyribonucleic acid (DNA) damage were assessed at different exposure times. A higher suppression in cell growth and viability was observed under a longer period of blue LED light exposure. The number of apoptotic cells increased as the light exposure time was prolonged. Reactive oxygen species (ROS) generation was also elevated in accordance to the extension of light exposure time. A comparison with dark‐maintained cells revealed that the upregulation of ROS by blue LED light plays a significant role in causing cellular dysfunction in DNA in a time‐dependent manner. In turn, antioxidant treatment has been shown to improve cell growth and viability under blue LED light conditions. This indicates that antioxidants have potential against blue LED light‐induced somatic cell damage. It is expected that this study will contribute to the understanding of the basic mechanism of somatic cell death under visible light and maximize the beneficial use of LED light in future animal experiments.     

Blue LED light exposure develops intracellular reactive oxygen species,lipid peroxidation,and subsequent cellular injuries in cultured bovine retinal pigment epithelial cells

Abstract

The effects of blue light emitter diode (LED) light exposure on retinal pigment epithelial cells (RPE cells) were examined to detect cellular damage or change and to clarify its mechanisms. The RPE cells were cultured and exposed by blue (470 nm) LED at 4.8 mW/cm². The cellular viability was determined by XTT assay and cellular injury was determined by the lactate dehydrogenase activity in medium. Intracellular reactive oxygen species (ROS) generation was determined by confocal laser microscope image analysis using dihydrorhodamine 123 and lipid peroxidation was determined by 4-hydroxy-2-nonenal protein-adducts immunofluorescent staining (HNE). At 24 h after 50 J/cm² exposures, cellular viability was significantly decreased to 74% and cellular injury was significantly increased to 365% of control. Immediately after the light exposure, ROS generation was significantly increased to 154%, 177%, and 395% of control and HNE intensity was increased to 211%, 359%, and 746% of control by 1, 10, and 50 J/cm², respectively. These results suggest, at least in part, that oxidative stress is an early step leading to cellular damage by blue LED exposure and cellular oxidative damage would be caused by the blue light exposure at even lower dose (1, 10 J/cm²).     

Icariside II,a novel phosphodiesterase 5 inhibitor,protects against H2O2‐induced PC12 cells death by inhibiting mitochondria‐mediated autophagy

Oxidative stress is a major cause of cellular injury in a variety of human diseases including neurodegenerative disorders. Thus, removal of excessive reactive oxygen species (ROS) or suppression of ROS generation may be effective in preventing oxidative stress‐induced cell death. This study was designed to investigate the effect of icariside II (ICS II), a novel phosphodiesterase 5 inhibitor, on hydrogen peroxide (H₂O₂)‐induced death of highly differentiated rat neuronal PC12 cells, and to further examine the underlying mechanisms. We found that ICS II pre‐treatment significantly abrogated H₂O₂‐induced PC12 cell death as demonstrated by the increase of the number of metabolically active cells and decrease of intracellular lactate dehydrogenase (LDH) release. Furthermore, ICS II inhibited H₂O₂‐induced cell death through attenuating intracellular ROS production, mitochondrial impairment, and activating glycogen synthase kinase‐3β (GSK‐3β) as demonstrated by reduced intracellular and mitochondrial ROS levels, restored mitochondrial membrane potential (MMP), decreased p‐tyr216‐GSK‐3β level and increased p‐ser9‐GSK‐3β level respectively. The GSK‐3β inhibitor SB216763 abrogated H₂O₂‐induced cell death. Moreover, ICS II significantly inhibited H₂O₂‐induced autophagy by the reducing autophagosomes number and the LC3‐II/LC3‐I ratio, down‐regulating Beclin‐1 expression, and up‐regulating p62/SQSTM1 and HSP60 expression. The autophagy inhibitor 3‐methyl adenine (3‐MA) blocked H₂O₂‐induced cell death. Altogether, this study demonstrated that ICS II may alleviate oxidative stress‐induced autophagy in PC12 cells, and the underlying mechanisms are related to its antioxidant activity functioning via ROS/GSK‐3β/mitochondrial signalling pathways.     

Studies on the relationship between pulsed UV light irradiation and the simultaneous occurrence of molecular and cellular damage in clinically-relevant Candida albicans

This constitutes the first study to report on the relationship between pulsed UV light (PL) irradiation and the simultaneous occurrence of molecular and cellular damage in clinical strains of Candida albicans. Microbial protein leakage and propidium iodide (PI) uptake assays demonstrated significant increases in cell membrane permeability in PL-treated yeast that depended on the amount of UV pulses applied. This finding correlated well with the measurement of increased levels of lipid hydroperoxidation in the cell membrane of PL-treated yeast. PL-treated yeast cells also displayed a specific pattern of intracellular reactive oxygen species (ROS) generation, where ROS were initially localised in the mitochondria after low levels of pulsing (UV dose 0.82 μJ/cm²) before more wide-spread cytosolic ROS production occurred with enhanced pulsing. Intracellular ROS levels were measured using the specific mitochondrial peroxide stain dihydrorhodamine 123 and the cytosolic oxidation stain dichloroflurescin diacetate. Use of the dihydroethidium stain also revealed increased levels of intracellular superoxide as a consequence of augmented pulsing. The ROS bursts observed during the initial phases of PL treatment was consistent with the occurrence of apoptotic cells as confirmed by detection of specific apoptotic markers, abnormal chromatin condensation and externalisation of cell membrane lipid phosphatidylserine. Increased amount of PL-irradiation (ca. UV does 1.24-1.65 μJ/cm²) also resulted in the occurrence of late apoptotic and necrotic yeast phenotypes, which coincided with the transition from mitochondrial to cytosolic localisation of ROS and with irreversible cell membrane leakage. Use of the comet assay also revealed significant nuclear damage in similarly treated PL samples. Although some level of cellular repair was observed in all test strains during sub-lethal exposure to PL-treatments (≤ 20 pulses or UV dose 0.55 μJ/cm²), this was absent in similar samples exposed to increased amounts of pulsing. This study showed that PL-irradiation inactivates C. albicans test strains through a multi-targeted process with no evidence of microbial ability to support cell growth after ≤ 20 pulses. Implications of our findings in terms of application of PL for contact-surface disinfection are discussed.     

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.     

Mitochondrial reactive oxygen species and nitric oxide-mediated cancer cell apoptosis in 2-butylamino-2-demethoxyhypocrellin B photodynamic treatment

Photodynamic therapy (PDT) is a novel and promising cancer treatment which employs a combination of a photosensitizing chemical and visible light to induce apoptosis in cancer cells. Singlet oxygen has been recognized as the main origin of oxidative stress in PDT. However, the precise mechanism of PDT-induced apoptosis is not well characterized, especially the dualistic role of nitric oxide (NO). To dissect the apoptosis pathways triggered by PDT, the intracellular free radicals in MCF-7 cells were investigated by examining a novel photosensitizer 2-butylamino-2-demethoxyhypocrellin B (2-BA-2-DMHB)-mediated PDT. It was found that exposure of the cells to 2-BA-2-DMHB and irradiation resulted in a significant increase of intracellular ROS in minutes, and then followed by cytoplasmic free calcium enhancement, mitochondrial nitric oxide synthase (mtNOS) activation, cytochrome c release, and apoptotic death. Scavengers of singlet oxygen or NO could attenuate PDT-induced cell viability loss, nucleus morphology changes, cytochrome c release, mitochondria swelling, and apo-apoptosis gene p53 and p21 mRNA levels. The results suggested that both ROS and NO played important roles in the apoptosis-induced by PDT.     

Calcium‐stimulated mitogen‐activated protein kinase activation elicits Bcl‐xL downregulation and Bak upregulation in notexin‐treated human neuroblastoma SK‐N‐SH cells

Notechis scutatus scutatus notexin induced apoptotic death of SK‐N‐SH cells accompanied with downregulation of Bcl‐xL, upregulation of Bak, mitochondrial depolarization, and ROS generation. Upon exposure to notexin, Ca²⁺‐mediated JNK and p38 MAPK activation were observed in SK‐N‐SH cells. Production of ROS was a downstream event followed by Ca²⁺‐mediated mitochondrial alteration. Notexin‐induced cell death, mitochondrial depolarization, and ROS generation were suppressed by SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor). Moreover, phospho‐p38 MAPK and phospho‐JNK were proved to be involved in Bcl‐xL degradation, and overexpression of Bcl‐xL attenuated the cytotoxic effect of notexin. Bak upregulation was elicited by p38 MAPK‐mediated ATF‐2 activation and JNK‐mediated c‐Jun activation. Suppression of Bak upregulation by ATF‐2 siRNA or c‐Jun siRNA attenuated notexin‐evoked mitochondrial depolarization and rescued viability of notexin‐treated cells. Taken together, our data indicate that notexin‐induced apoptotic death of SK‐N‐SH cells is mediated through mitochondrial alteration triggering by Ca²⁺‐evoked p38 MAPK/ATF‐2 and JNK/c‐Jun signaling pathways. J. Cell. Physiol. 222:177–186, 2010. © 2009 Wiley‐Liss, Inc.     

Different pathways lead to mitochondrial fragmentation during apoptotic and excitotoxic cell death in primary neurons

Mitochondrial fragmentation is recognized to be an important event during the onset of apoptosis. In this current study, we have used single cell imaging to investigate the role of the mitochondrial fission protein DRP‐1 on mitochondrial morphology and mitochondrial fragmentation in primary hippocampal neurons undergoing necrotic or apoptotic cell death. Treatment of neurons with 500 nM staurosporine (apoptosis) or 30 μM glutamate (l ‐Glu; excitotoxic necrosis) produced a fragmentation and condensation of mitochondria, which although occurred over markedly different time frames appeared broadly similar in appearance. In neurons exposed to an apoptotic stimuli, inhibiting DRP‐1 activity using overexpression of the dominant negative DRP‐1^K38A slowed the rate of mitochondrial fragmentation and decreased total cell death when compared to overexpression of wild‐type DRP‐1. In contrast, responses to l ‐Glu appeared DRP‐1 independent. Similarly, alterations in the fission/fusion state of the mitochondrial network did not alter mitochondrial Ca²⁺ uptake or the ability of l ‐Glu to stimulate excitotoxic Ca²⁺ overload. Finally, apoptosis‐induced mitochondrial fragmentation was observed concurrent with recruitment of Bax to the mitochondrial membrane. In contrast, during glutamate excitotoxicity, Bax remained in the cytosolic compartment. We conclude that different pathways lead to the appearance of fragmented mitochondria during necrotic and apoptotic neuronal cell death. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:335–341, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20336     

Low‐level laser therapy (810 nm) protects primary cortical neurons against excitotoxicity in vitro

Excitotoxicity describes a pathogenic process whereby death of neurons releases large amounts of the excitatory neurotransmitter glutamate, which then proceeds to activate a set of glutamatergic receptors on neighboring neurons (glutamate, N‐methyl‐D‐aspartate (NMDA), and kainate), opening ion channels leading to an influx of calcium ions producing mitochondrial dysfunction and cell death. Excitotoxicity contributes to brain damage after stroke, traumatic brain injury, and neurodegenerative diseases, and is also involved in spinal cord injury. We tested whether low level laser (light) therapy (LLLT) at 810 nm could protect primary murine cultured cortical neurons against excitotoxicity in vitro produced by addition of glutamate, NMDA or kainate. Although the prevention of cell death was modest but significant, LLLT (3 J/cm² delivered at 25 mW/cm² over 2 min) gave highly significant benefits in increasing ATP, raising mitochondrial membrane potential, reducing intracellular calcium concentrations, reducing oxidative stress and reducing nitric oxide. The action of LLLT in abrogating excitotoxicity may play a role in explaining its beneficial effects in diverse central nervous system pathologies. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Adenoviral SERCA1 overexpression triggers an apoptotic response in cultured neonatal but not in adult rat cardiomyocytes

Although apoptosis and necrosis have been considered different pathways to cell death, only one compound induces both types of cell death. Diethyldithiocarbamate (DDC) has been shown to have antioxidant or prooxidant effects in several different systems. We observed in our present study that DDC induced not only apoptosis but also necrosis depending on its dosage in HL60 premyelocytic leukemia cells. Moreover, in hypoxia cell culture conditions, DDC-induced necrotic cells decreased but DDC-induced apoptosis continued. We investigated the DDC-induced different cell death mechanisms as they are correlated with reactive oxygen species (ROS). High-dose DDC-induced necrotic cell death is thought to depend on the increase of intracellular ROS, while low-dose DDC-induced apoptosis is thought to depend on changes of the intracellular redox state by the transporting of external metal ions. There was no sequential or quantitative change of Bcl-2 family proteins in DDC-induced apoptotic or necrotic pathways. However, the mitochondrial transmembrane potential was remarkably decreased in the DDC-induced necrosis. Finally, duration of c-Jun N-terminal kinase (JNK) activation resulted in different types of cell death.     

Light‐emitting diode irradiation induces AKT/mTOR‐mediated apoptosis in human pancreatic cancer cells and xenograft mouse model

The beneficial effects of light‐emitting diode (LED) irradiation have been reported in various pathologies, including cancer. However, its effect in pancreatic cancer cells remains unclear. Herein, we demonstrated that blue LED of 460 nm regulated pancreatic cancer cell proliferation and apoptosis by suppressing the expression of apoptosis‐related factors, such as mutant p53 and B‐cell lymphoma 2 (Bcl‐2), and decreasing the expression of RAC‐β serine/threonine kinase 2 (AKT2), the phosphorylation of protein kinase B (AKT), and mammalian target of rapamycin (mTOR). Blue LED irradiation also increased the levels of cleaved poly‐(ADP‐ribose) polymerase (PARP) and caspase‐3 in pancreatic cancer cells, while it suppressed AKT2 expression and inhibited tumor growth in xenograft tumor tissues. In conclusion, blue LED irradiation suppressed pancreatic cancer cell and tumor growth by regulating AKT/mTOR signaling. Our findings indicated that blue LEDs could be used as a nonpharmacological treatment for pancreatic cancer.     

Chemical Hypoxia-Induced Cell Death in Human Glioma Cells: Role of Reactive Oxygen Species,ATP Depletion,Mitochondrial Damage and Ca2+

This study was undertaken to evaluate whether chemical hypoxia-induced cell injury is a result of reactive oxygen species (ROS) generation, ATP depletion, mitochondrial permeability transition, and an increase in intracellular Ca²⁺, in A172 cells, a human glioma cell line. Chemical hypoxia was induced by incubating cells with antimycin A, an inhibitor of mitochondrial electron transport, in a glucose-free medium. Exposure of cells to chemical hypoxia resulted in cell death, ROS generation, ATP depletion, and mitochondrial permeability transition. The H₂O₂ scavenger pyruvate prevented cell death, ROS generation, and mitochondrial permeability transition induced by chemical hypoxia. In contrast, changes mediated by chemical hypoxia were not affected by hydroxyl radical scavengers. Antioxidants did not affect cell death and ATP depletion induced by chemical hypoxia, although they prevented ROS production and mitochondrial permeability transition induced by chemical hypoxia. Chemical hypoxia did not increase lipid peroxidation even when antimycin A was increased to 50 M, whereas the oxidant t-butylhydroperoxide caused a significant increase in lipid peroxidation, at a concentration that is less effective than chemical hypoxia in inducing cell death. Fructose protected against cell death and mitochondrial permeability transition induced by chemical hypoxia. However, ROS generation and ATP depletion were not prevented by fructose. Chemical hypoxia caused the early increase in intracellular Ca²⁺. The cell death and ROS generation induced by chemical hypoxia were altered by modulation of intracellular Ca²⁺ concentration with ruthenium red, TMB-8, and BAPTA/AM. However, mitochondrial permeability transition was not affected by these compounds. These results indicate that chemical hypoxia causes cell death, which may be, in part, mediated by H₂O₂ generation via a lipid peroxidation-independent mechanism and elevated intracellular Ca²⁺. In addition, these data suggest that chemical hypoxia-induced cell death is not associated directly with ATP depletion and mitochondrial permeability transition.