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1.
J Dermatol Sci ; 114(1): 24-33, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38448340

ABSTRACT

BACKGROUND: The unfolded protein response (UPR) is one of the cytoprotective mechanisms against various stresses and essential for the normal function of skin. Skin injury caused by ionizing radiation (IR) is a common side effect of radiotherapy and it is unclear how UPR affects IR-induced skin injury. OBJECTIVES: To verify the effect of UPR on IR-induced DNA damage in keratinocytes and the relation between an endoplasmic reticulum (ER) protein KTN1 and UPR. METHODS: All experiments were performed on keratinocytes models: HaCaT and HEK-A. ER lumen and the expression levels of KTN1 and UPR pathway proteins (PERK, IRE1α and ATF6) were examined by transmission electron microscopy and immunoblotting, respectively. 4-PBA, an UPR inhibitor, was used to detected its effects on DNA damage and cell proliferation. Subsequently, the effects of KTN1 deletion on UPR, DNA damage and cell proliferation after IR were detected. Tunicamycin was used to reactivate UPR and then we examined its effects on DNA damage. RESULTS: UPR was activated by IR in keratinocytes. Inhibition of UPR aggravated DNA damage and suppressed cell proliferation after IR. KTN1 expression was upregulated by IR and KTN1 depletion reduced ER expansion and the expression of UPR-related proteins. Moreover, KTN1 depletion aggravated DNA damage and suppressed cell proliferation after IR could reversed by reactivation of UPR. CONCLUSION: KTN1 deletion aggravates IR-induced keratinocyte DNA damage via inhibiting UPR. Our findings provide new insights into the mechanisms of keratinocytes in response to IR-induced damage.


Subject(s)
Cell Proliferation , DNA Damage , HaCaT Cells , Keratinocytes , Radiation, Ionizing , Unfolded Protein Response , Humans , Cell Line , Cell Proliferation/radiation effects , Cell Proliferation/drug effects , DNA Damage/radiation effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/radiation effects , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum Stress/radiation effects , Endoplasmic Reticulum Stress/drug effects , Keratinocytes/radiation effects , Keratinocytes/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , Skin/radiation effects , Skin/pathology , Skin/cytology , Skin/drug effects , Skin/metabolism , Unfolded Protein Response/radiation effects , Unfolded Protein Response/drug effects
2.
J Photochem Photobiol B ; 214: 112088, 2021 Jan.
Article in English | MEDLINE | ID: mdl-33278762

ABSTRACT

Low level light therapy uses light of specific wavelengths in red and near-infrared spectral range to treat various pathological conditions. This light is able to modulate biochemical cascade reactions in cells that can have important health implications. In this study, the effect of low intensity light at 650, 808 and 1064 nm on neurons and two types of cancer cells (neuroblastoma and HeLa) is reported, with focus on the photoinduced change of intracellular level of Ca2+ ions and corresponding signaling pathways. The obtained results show that 650 and 808 nm light promotes intracellular Ca2+ elevation regardless of cell type, but with different dynamics due to the specificities of Ca2+ regulation in neurons and cancer cells. Two origins responsible for Ca2+ elevation are determined to be: influx of exogenous Ca2+ ions into cells and Ca2+ release from endoplasmic reticulum. Our investigation of the related cellular processes shows that light-induced membrane depolarization is distinctly involved in the mechanism of Ca2+ influx. Ca2+ release from endoplasmic reticulum activated by reactive oxygen species generation is considered as a possible light-dependent signaling pathway. In contrast to the irradiation with 650 and 808 nm light, no effects are observed under 1064 nm irradiation. We believe that the obtained insights are of high significance and can be useful for the development of drug-free phototherapy.


Subject(s)
Calcium Signaling/radiation effects , Calcium/radiation effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/radiation effects , Calcium/physiology , Cell Membrane/metabolism , Electrophysiology , Fluorescent Dyes/chemistry , HeLa Cells , Humans , Infrared Rays , Low-Level Light Therapy , Neurons/radiation effects , Optical Imaging , Reactive Oxygen Species/radiation effects
3.
Mol Plant ; 13(11): 1545-1555, 2020 11 02.
Article in English | MEDLINE | ID: mdl-32992028

ABSTRACT

Plants often encounter light intensities exceeding the capacity of photosynthesis (excessive light) mainly due to biotic and abiotic factors, which lower CO2 fixation and reduce light energy sinks. Under excessive light, the photosynthetic electron transport chain generates damaging molecules, hence leading to photooxidative stress and eventually to cell death. In this review, we summarize the mechanisms linking the excessive absorption of light energy in chloroplasts to programmed cell death in plant leaves. We highlight the importance of reactive carbonyl species generated by lipid photooxidation, their detoxification, and the integrating role of the endoplasmic reticulum in the adoption of phototolerance or cell-death pathways. Finally, we invite the scientific community to standardize the conditions of excessive light treatments.


Subject(s)
Light/adverse effects , Plant Cells/radiation effects , Apoptosis/radiation effects , Chloroplasts/radiation effects , Endoplasmic Reticulum/radiation effects , Lipid Peroxidation/radiation effects , Plant Growth Regulators/physiology , Plant Leaves/cytology , Plant Leaves/radiation effects
4.
J Dermatol Sci ; 98(1): 41-49, 2020 Apr.
Article in English | MEDLINE | ID: mdl-32376153

ABSTRACT

BACKGROUND: Endoplasmic reticulum (ER) calcium depletion-induced ER stress is a crucial signal for keratinocyte differentiation and barrier homeostasis, but its effects on the epidermal tight junction (TJ) have not been characterized. Ultraviolet B (UVB) causes ER calcium release in keratinocytes and disrupts epidermal TJ, however, the involvement of ER stress in the UVB-induced TJ alterations remains unknown. OBJECTIVES: To investigate the effect of ER stress by pharmacological ER calcium depletion or UVB on the TJ integrity in normal human epidermal keratinocytes (NHEK). METHODS: NHEK were exposed to ER calcium pump inhibitor thapsigargin (Tg) or UVB. ER stress markers and TJ molecules expression, TJ and F-actin structures, and TJ barrier function were analyzed. RESULTS: Tg or UVB exposure dose-dependently triggered unfolded protein response (UPR) in NHEK. Low dose Tg induced the IRE1α-XBP1 pathway and strengthened TJ barrier. Contrary, high dose Tg activated PERK phosphorylation and disrupted TJ by F-actin disorganization. UVB disrupted TJ and F-actin structures dose dependently. IRE1α RNase inhibition induced or exacerbated TJ and F-actin disruption in the presence of low dose Tg or UVB. High dose Tg increased RhoA activity. 4-PBA or Rho kinase (ROCK) inhibitor partially prevented the disruption of TJ and F-actin following high dose Tg or UVB. CONCLUSIONS: ER stress has bimodal effects on the epidermal TJ depending on its intensity. The IRE1α pathway is critical for the maintenance of TJ integrity during mild ER stress. Severe ER stress-induced UPR or ROCK signalling mediates the disruption of TJ through cytoskeletal disorganization during severe ER stress.


Subject(s)
Calcium/metabolism , Endoplasmic Reticulum Stress/radiation effects , Keratinocytes/pathology , Tight Junctions/pathology , Ultraviolet Rays/adverse effects , Amides/pharmacology , Cell Line , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/pathology , Endoplasmic Reticulum/radiation effects , Endoplasmic Reticulum Stress/drug effects , Endoribonucleases/metabolism , Humans , Keratinocytes/cytology , Keratinocytes/radiation effects , Phenylbutyrates/pharmacology , Protein Serine-Threonine Kinases/metabolism , Pyridines/pharmacology , Signal Transduction/drug effects , Signal Transduction/radiation effects , Tight Junctions/drug effects , Tight Junctions/metabolism , Tight Junctions/radiation effects , Unfolded Protein Response/drug effects , Unfolded Protein Response/radiation effects , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/metabolism
5.
Biochem Biophys Res Commun ; 524(4): 869-875, 2020 04 16.
Article in English | MEDLINE | ID: mdl-32051089

ABSTRACT

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a neuroprotective factor produced in response to endoplasmic reticulum (ER) stress induced by various stressors, but its involvement in the radioresistance of tumor cells is unknown. Here, we found that MANF is released after γ-irradiation (2 Gy and 4 Gy) of B16 melanoma cells, and its release was suppressed by 4-phenylbutyric acid, an ER stress inhibitor. MANF was not released after low-dose (1 Gy) γ-irradiation, but pretreatment of 1 Gy-irradiated cells with recombinant MANF enhanced the cellular DNA damage response and attenuated reproductive cell death. In MANF-knockdown cells, the DNA damage response and p53 activation by γ-irradiation (2 Gy) were suppressed, and reproductive cell death was increased. MANF also activated the ERK signaling pathway. Our findings raise the possibility that MANF could be a new target for overcoming radioresistance.


Subject(s)
Endoplasmic Reticulum Stress/radiation effects , Endoplasmic Reticulum/radiation effects , Gene Expression Regulation, Neoplastic , Nerve Growth Factors/genetics , Radiation Tolerance/genetics , Animals , Cell Line, Tumor , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum Stress/genetics , Gamma Rays , Melanoma, Experimental/genetics , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Melanoma, Experimental/radiotherapy , Mice , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Nerve Growth Factors/antagonists & inhibitors , Nerve Growth Factors/metabolism , Phenylbutyrates/pharmacology , Phosphorylation , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
6.
Plant Cell Rep ; 39(4): 473-487, 2020 Apr.
Article in English | MEDLINE | ID: mdl-32016506

ABSTRACT

KEY MESSAGE: The non-intrinsic ABC proteins ABCI20 and ABCI21 are induced by light under HY5 regulation, localize to the ER, and ameliorate cytokinin-driven growth inhibition in young Arabidopsis thaliana seedlings. The plant ATP-binding cassette (ABC) I subfamily (ABCIs) comprises heterogeneous proteins containing any of the domains found in other ABC proteins. Some ABCIs are known to function in basic metabolism and stress responses, but many remain functionally uncharacterized. ABCI19, ABCI20, and ABCI21 of Arabidopsis thaliana cluster together in a phylogenetic tree, and are suggested to be targets of the transcription factor ELONGATED HYPOCOTYL 5 (HY5). Here, we reveal that these three ABCIs are involved in modulating cytokinin responses during early seedling development. The ABCI19, ABCI20 and ABCI21 promoters harbor HY5-binding motifs, and ABCI20 and ABCI21 expression was induced by light in a HY5-dependent manner. abci19 abci20 abci21 triple and abci20 abci21 double knockout mutants were hypersensitive to cytokinin in seedling growth retardation assays, but did not show phenotypic differences from the wild type in either control medium or auxin-, ABA-, GA-, ACC- or BR-containing media. ABCI19, ABCI20, and ABCI21 were expressed in young seedlings and the three proteins interacted with each other, forming a large protein complex at the endoplasmic reticulum (ER) membrane. These results suggest that ABCI19, ABCI20, and ABCI21 fine-tune the cytokinin response at the ER under the control of HY5 at the young seedling stage.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , Cytokinins/metabolism , Endoplasmic Reticulum/metabolism , ATP-Binding Cassette Transporters/genetics , Amino Acid Motifs , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis/radiation effects , Arabidopsis Proteins/genetics , Basic-Leucine Zipper Transcription Factors/genetics , Cytokinins/genetics , Endoplasmic Reticulum/radiation effects , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/radiation effects , Gene Knockout Techniques , Light , Phylogeny , Plants, Genetically Modified , Promoter Regions, Genetic , Protein Binding , Seedlings/genetics , Seedlings/growth & development , Seedlings/metabolism , Seedlings/radiation effects
7.
Radiat Res ; 193(1): 63-72, 2020 01.
Article in English | MEDLINE | ID: mdl-31714866

ABSTRACT

It is well known that mitochondria and the endoplasmic reticulum (ER) play important roles in radiation response, but their functions in radiation-induced bystander effect (RIBE) are largely unclear. In this study, we found that when a small portion of cells in a population of human lung fibroblast MRC-5 cells were precisely irradiated through either the nuclei or cytoplasm with counted microbeam protons, the yield of micronuclei (MN) and the levels of intracellular reactive oxygen species (ROS) in nonirradiated cells neighboring irradiated cells were significantly increased. Mito/ER-tracker staining demonstrated that the mitochondria were clearly activated after nuclear irradiation and ER mass approached a higher level after cytoplasmic irradiation. Moreover, the radiation-induced ROS was diminished by rotenone, an inhibitor of mitochondria activation, but it was not influenced by siRNA interference of BiP, an ER regulation protein. While for nuclear irradiation, rotenone-enhanced radiation-induced ER expression, and BiP siRNA eliminated radiation-induced activation of mitochondria, these phenomena were not observed for cytoplasmic irradiation. Bystander MN was reduced by rotenone but enhanced by BiP siRNA. When the cells were treated with both rotenone and BiP siRNA, the MN yield was reduced for nuclear irradiation but was enhanced for cytoplasmic irradiation. Our results suggest that the organelles of mitochondria and ER have different roles in RIBE with respect to nuclear and cytoplasmic irradiation, and the function of ER is a prerequisite for mitochondrial activation.


Subject(s)
Bystander Effect/radiation effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/radiation effects , Mitochondria/metabolism , Mitochondria/radiation effects , Protons/adverse effects , Cell Line , Fibroblasts/cytology , Fibroblasts/radiation effects , Humans , Reactive Oxygen Species/metabolism
8.
Free Radic Biol Med ; 131: 27-39, 2019 02 01.
Article in English | MEDLINE | ID: mdl-30496813

ABSTRACT

Today the noxiousness of blue light from natural and particularly artificial (fluorescent tubes, LED panels, visual displays) sources is actively discussed in the context of various ocular diseases. Many of them have an important neurologic component and are associated with ocular pain. This neuropathic signal is provided by nociceptive neurons from trigeminal ganglia. However, the phototoxicity of blue light on trigeminal neurons has not been explored so far. The aim of the present in vitro study was to investigate the cytotoxic impact of various wavebands of visible light (410-630 nm) on primary cell culture of mouse trigeminal neural and glial cells. Three-hour exposure to narrow wavebands of blue light centered at 410, 440 and 480 nm of average 1.1 mW/cm2 irradiance provoked cell death, altered cell morphology and induced oxidative stress and inflammation. These effects were not observed for other tested visible wavebands. We observed that neurons and glial cells processed the light signal in different manner, in terms of resulting superoxide and hydrogen peroxide generation, inflammatory biomarkers expression and phototoxic mitochondrial damage. We analyzed the pathways of photic signal reception, and we proposed that, in trigeminal cells, in addition to widely known mitochondria-mediated light absorption, light could be received by means of non-visual opsins, melanopsin (opn4) and neuropsin (opn5). We also investigated the mechanisms underlying the observed phototoxicity, further suggesting an important role of the endoplasmic reticulum in neuronal transmission of blue-light-toxic message. Taken together, our results give some insight into circuit of tangled pain and photosensitivity frequently observed in patients consulting for these ocular symptoms.


Subject(s)
Cell Death/radiation effects , Hydrogen Peroxide/agonists , Light/adverse effects , Neuroglia/radiation effects , Neurons/radiation effects , Superoxides/agonists , Animals , Dose-Response Relationship, Radiation , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/radiation effects , Gene Expression/radiation effects , Hydrogen Peroxide/metabolism , Light Signal Transduction , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mitochondria/metabolism , Mitochondria/radiation effects , Neuroglia/metabolism , Neurons/metabolism , Opsins/genetics , Opsins/metabolism , Oxidative Stress/radiation effects , Primary Cell Culture , Rod Opsins/genetics , Rod Opsins/metabolism , Superoxides/metabolism , Trigeminal Ganglion/metabolism , Trigeminal Ganglion/radiation effects
9.
Biomed Environ Sci ; 31(8): 561-571, 2018 Aug.
Article in English | MEDLINE | ID: mdl-30231960

ABSTRACT

OBJECTIVE: To detect the effects of microwave on calcium levels in primary hippocampal neurons and primary cardiomyocytes by the real-time microwave exposure combined with laser scanning confocal microscopy. METHODS: The primary hippocampal neurons and primary cardiomyocytes were cultured and labeled with probes, including Fluo-4 AM, Mag-Fluo-AM, and Rhod-2, to reflect the levels of whole calcium [Ca2+], endoplasmic reticulum calcium [Ca2+]ER, and mitochondrial calcium [Ca2+]MIT, respectively. Then, the cells were exposed to a pulsed microwave of 2.856 GHz with specific absorption rate (SAR) values of 0, 4, and 40 W/kg for 6 min to observe the changes in calcium levels. RESULTS: The results showed that the 4 and 40 W/kg microwave radiation caused a significant decrease in the levels of [Ca2+], [Ca2+]ER, and [Ca2+]MIT in primary hippocampal neurons. In the primary cardiomyocytes, only the 40 W/kg microwave radiation caused the decrease in the levels of [Ca2+], [Ca2+]ER, and [Ca2+]MIT. Primary hippocampal neurons were more sensitive to microwave exposure than primary cardiomyocytes. The mitochondria were more sensitive to microwave exposure than the endoplasmic reticulum. CONCLUSION: The calcium efflux was occurred during microwave exposure in primary hippocampal neurons and primary cardiomyocytes. Additionally, neurons and mitochondria were sensitive cells and organelle respectively.


Subject(s)
Calcium/metabolism , Microwaves , Myocytes, Cardiac/radiation effects , Neurons/radiation effects , Animals , Cells, Cultured , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/radiation effects , Hippocampus/cytology , Mitochondria/metabolism , Mitochondria/radiation effects , Myocytes, Cardiac/metabolism , Neurons/metabolism , Rats, Wistar
10.
Mol Cancer Res ; 16(10): 1447-1453, 2018 10.
Article in English | MEDLINE | ID: mdl-29991528

ABSTRACT

The aggressive nature and inherent therapeutic resistance of glioblastoma multiforme (GBM) has rendered the median survival of afflicted patients to 14 months. Therefore, it is imperative to understand the molecular biology of GBM to provide new treatment options to overcome this disease. It has been demonstrated that the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway is an important regulator of the endoplasmic reticulum (ER) stress response. PERK signaling has been observed in other model systems after radiation; however, less is known in the context of GBM, which is frequently treated with radiation-based therapies. To investigate the significance of PERK, we studied activation of the PERK-eIF2α-ATF4 pathway in GBM after ionizing radiation (IR). By inhibiting PERK, it was determined that ionizing radiation (IR)-induced PERK activity led to eIF2α phosphorylation. IR enhanced the prodeath component of PERK signaling in cells treated with Sal003, an inhibitor of phospho-eIF2α phosphatase. Mechanistically, ATF4 mediated the prosurvival activity during the radiation response. The data support the notion that induction of ER stress signaling by radiation contributes to adaptive survival mechanisms during radiotherapy. The data also support a potential role for the PERK/eIF2α/ATF4 axis in modulating cell viability in irradiated GBM.Implications: The dual function of PERK as a mediator of survival and death may be exploited to enhance the efficacy of radiation therapy.Visual Overview: http://mcr.aacrjournals.org/content/16/10/1447/F1.large.jpg Mol Cancer Res; 16(10); 1447-53. ©2018 AACR.


Subject(s)
Activating Transcription Factor 4/genetics , Eukaryotic Initiation Factor-2/genetics , Glioblastoma/radiotherapy , Radiation Tolerance/genetics , eIF-2 Kinase/genetics , Cell Line, Tumor , Cell Proliferation/radiation effects , Endoplasmic Reticulum/radiation effects , Endoplasmic Reticulum Stress/genetics , Eukaryotic Initiation Factor-2/antagonists & inhibitors , Gene Expression Regulation, Neoplastic/radiation effects , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Lentivirus/genetics , Phosphorylation/radiation effects , Radiation, Ionizing , Signal Transduction/radiation effects , Transfection
11.
J Neurophysiol ; 120(2): 509-524, 2018 08 01.
Article in English | MEDLINE | ID: mdl-29668377

ABSTRACT

Inner ear spiral ganglion neurons were cultured from day 4 postnatal mice and loaded with a fluorescent Ca2+ indicator (fluo-4, -5F, or -5N). Pulses of infrared radiation (IR; 1,863 nm, 200 µs, 200-250 Hz for 2-5 s, delivered via an optical fiber) produced a rapid, transient temperature increase of 6-12°C (above a baseline of 24-30°C). These IR pulse trains evoked transient increases in both nuclear and cytosolic Ca2+ concentration ([Ca2+]) of 0.20-1.4 µM, with a simultaneous reduction of [Ca2+] in regions containing endoplasmic reticulum (ER). IR-induced increases in cytosolic [Ca2+] continued in medium containing no added Ca2+ (±Ca2+ buffers) and low [Na+], indicating that the [Ca2+] increase was mediated by release from intracellular stores. Consistent with this hypothesis, the IR-induced [Ca2+] response was prolonged and eventually blocked by inhibition of ER Ca2+-ATPase with cyclopiazonic acid, and was also inhibited by a high concentration of ryanodine and by inhibitors of inositol (1,4,5)-trisphosphate (IP3)-mediated Ca2+ release (xestospongin C and 2-aminoethoxydiphenyl borate). The thermal sensitivity of the response suggested involvement of warmth-sensitive transient receptor potential (TRP) channels. The IR-induced [Ca2+] increase was inhibited by TRPV4 inhibitors (HC-067047 and GSK-2193874), and immunostaining of spiral ganglion cultures demonstrated the presence of TRPV4 and TRPM2 that colocalized with ER marker GRP78. These results suggest that the temperature sensitivity of IR-induced [Ca2+] elevations is conferred by TRP channels on ER membranes, which facilitate Ca2+ efflux into the cytosol and thereby contribute to Ca2+-induced Ca2+-release via IP3 and ryanodine receptors. NEW & NOTEWORTHY Infrared radiation-induced photothermal effects release Ca2+ from the endoplasmic reticulum of primary spiral ganglion neurons. This Ca2+ release is mediated by activation of transient receptor potential (TRPV4) channels and involves amplification by Ca2+-induced Ca2+-release. The neurons immunostained for warmth-sensitive channels, TRPV4 and TRPM2, which colocalize with endoplasmic reticulum. Pulsed infrared radiation provides a novel experimental tool for releasing intracellular Ca2+, studying Ca2+ regulatory mechanisms, and influencing neuronal excitability.


Subject(s)
Calcium/metabolism , Endoplasmic Reticulum/metabolism , Hot Temperature , Infrared Rays , Neurons/metabolism , Spiral Ganglion/metabolism , TRPV Cation Channels/metabolism , Animals , Cells, Cultured , Endoplasmic Reticulum/radiation effects , Endoplasmic Reticulum Chaperone BiP , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Mice, Inbred C57BL , Neurons/radiation effects , Ryanodine Receptor Calcium Release Channel/metabolism , Spiral Ganglion/radiation effects , TRPM Cation Channels/metabolism
12.
J Med Chem ; 61(9): 3952-3961, 2018 05 10.
Article in English | MEDLINE | ID: mdl-29681157

ABSTRACT

Two advanced boron dipyrromethene (BODIPY) based photosensitizers have been synthesized and characterized. With a glibenclamide analogous moiety, these compounds can localize in the endoplasmic reticulum (ER) of HeLa human cervical carcinoma cells and HepG2 human hepatocarcinoma cells. The BODIPY π skeleton is conjugated with two styryl or carbazolylethenyl groups, which can substantially red-shift the Q-band absorption and fluorescence emission and impart two-photon absorption (TPA) property to the chromophores. The TPA cross section of the carbazole-containing analogue reaches a value of 453 GM at 1010 nm. These compounds also behave as singlet oxygen generators with high photostability. Upon irradiation at λ > 610 nm, these photosensitizers cause photocytotoxicity to these two cell lines with IC50 values down to 0.09 µM, for which the cell death is triggered mainly by ER stress. The two-photon photodynamic activity of the distyryl derivative upon excitation at λ = 800 nm has also been demonstrated.


Subject(s)
Boron/chemistry , Endoplasmic Reticulum/drug effects , Photochemotherapy , Photons , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacology , Porphobilinogen/analogs & derivatives , Absorption, Radiation , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/radiation effects , HeLa Cells , Hep G2 Cells , Humans , Porphobilinogen/chemistry , Reactive Oxygen Species/metabolism , Singlet Oxygen/metabolism , Structure-Activity Relationship
13.
Sci Rep ; 7(1): 13079, 2017 10 12.
Article in English | MEDLINE | ID: mdl-29026094

ABSTRACT

Microsecond pulsed electric fields (µsPEF) permeabilize the plasma membrane (PM) and are widely used in research, medicine and biotechnology. For internal membranes permeabilization, nanosecond pulsed electric fields (nsPEF) are applied but this technology is complex to use. Here we report that the endoplasmic reticulum (ER) membrane can also be electropermeabilized by one 100 µs pulse without affecting the cell viability. Indeed, using Ca2+ as a permeabilization marker, we observed cytosolic Ca2+ peaks in two different cell types after one 100 µs pulse in a medium without Ca2+. Thapsigargin abolished these Ca2+ peaks demonstrating that the calcium is released from the ER. Moreover, IP3R and RyR inhibitors did not modify these peaks showing that they are due to the electropermeabilization of the ER membrane and not to ER Ca2+ channels activation. Finally, the comparison of the two cell types suggests that the PM and the ER permeabilization thresholds are affected by the sizes of the cell and the ER. In conclusion, this study demonstrates that µsPEF, which are easier to control than nsPEF, can permeabilize internal membranes. Besides, µsPEF interaction with either the PM or ER, can be an efficient tool to modulate the cytosolic calcium concentration and study Ca2+ roles in cell physiology.


Subject(s)
Calcium/metabolism , Cell Membrane/metabolism , Cell Membrane/radiation effects , Electroporation/methods , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/radiation effects , Animals , Calcium Channels/metabolism , Cell Line , Cell Survival/physiology , Cricetulus , Humans , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism
14.
Cell Calcium ; 63: 43-47, 2017 05.
Article in English | MEDLINE | ID: mdl-28108028

ABSTRACT

The inositol trisphosphate (IP3) signaling pathway evokes local Ca2+ signals (Ca2+ puffs) that arise from the concerted openings of clustered IP3 receptor/channels in the ER membrane. Physiological activation is triggered by binding of agonists to G-protein-coupled receptors (GPCRs) on the cell surface, leading to cleavage of phosphatidyl inositol bisphosphate and release of IP3 into the cytosol. Photorelease of IP3 from a caged precursor provides a convenient and widely employed means to study the final stage of IP3-mediated Ca2+ liberation, bypassing upstream signaling events to enable more precise control of the timing and relative concentration of cytosolic IP3. Here, we address whether Ca2+ puffs evoked by photoreleased IP3 fully replicate those arising from physiological agonist stimulation. We imaged puffs in individual SH-SY5Y neuroblastoma cells that were sequentially stimulated by picospritzing extracellular agonist (carbachol, CCH or bradykinin, BK) followed by photorelease of a poorly-metabolized IP3 analog, i-IP3. The centroid localizations of fluorescence signals during puffs evoked in the same cells by agonists and photorelease substantially overlapped (within ∼1µm), suggesting that IP3 from both sources accesses the same, or closely co-localized clusters of IP3Rs. Moreover, the time course and spatial spread of puffs evoked by agonists and photorelease matched closely. Because photolysis generates IP3 uniformly throughout the cytoplasm, our results imply that IP3 generated in SH-SY5Y cells by activation of receptors to CCH and BK also exerts broadly distributed actions, rather than specifically activating a subpopulation of IP3Rs that are scaffolded in close proximity to cell surface receptors to form a signaling nanodomain.


Subject(s)
Bradykinin/pharmacology , Calcium Signaling/drug effects , Calcium/metabolism , Carbachol/pharmacology , Inositol 1,4,5-Trisphosphate/metabolism , Light , Neuroblastoma/metabolism , Cholinergic Agonists/pharmacology , Cytosol/drug effects , Cytosol/metabolism , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/radiation effects , Humans , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Kinetics , Neuroblastoma/drug therapy , Neuroblastoma/pathology , Tumor Cells, Cultured , Vasodilator Agents/pharmacology
15.
Microsc Res Tech ; 80(5): 444-455, 2017 May.
Article in English | MEDLINE | ID: mdl-28094894

ABSTRACT

Several new features of mitochondrial nucleoid and its surroundings in mammalian cells were described previously (Prachar, 2016). Very small details were observed using the improved transmission electron microscopy method, as described in the article. In the meantime, the method has again been improved to 2 Å resolutions in the cell section. The method described in detail in the present work is documented on the same records that were published in lower resolution in the work Prachar (2016), enabling comparison of the achieved resolution with the previous one. New records are also presented, showing extremely high resolution and thus implying the importance of the method. Potential use of this method in different fields is suggested.


Subject(s)
Cells/ultrastructure , Microscopy, Electron, Transmission/methods , Radiation Exposure/prevention & control , Animals , Cell Line, Tumor , Cells/radiation effects , Electrons , Endoplasmic Reticulum/radiation effects , Endoplasmic Reticulum/ultrastructure , Epoxy Resins , Histological Techniques , Leukemia L1210 , Mice , Mitochondria/radiation effects , Mitochondria/ultrastructure , Plastic Embedding/standards , Ribosomes/radiation effects , Ribosomes/ultrastructure , Time Factors
16.
Oncotarget ; 7(48): 79885-79900, 2016 Nov 29.
Article in English | MEDLINE | ID: mdl-27829218

ABSTRACT

2-fluoro-2-deoxy-D-glucose (FDG), labeled with 18F radioisotope, is the most common imaging agent used for positron emission tomography (PET) in oncology. However, little is known about the cellular effects of FDG. Another glucose analogue, 2-deoxy-D-glucose (2DG), has been shown to affect many cellular functions, including intracellular transport and lipid metabolism, and has been found to improve the efficacy of cancer chemotherapeutic agents in vivo. Thus, in the present study, we have investigated cellular effects of FDG with the focus on changes in cellular lipids and intracellular transport. By quantifying more than 200 lipids from 17 different lipid classes in HEp-2 cells and by analyzing glycosphingolipids from MCF-7, HT-29 and HBMEC cells, we have discovered that FDG treatment inhibits glucosylceramide synthesis and thus reduces cellular levels of glycosphingolipids. In addition, in HEp-2 cells the levels and/or species composition of other lipid classes, namely diacylglycerols, phosphatidic acids and phosphatidylinositols, were found to change upon treatment with FDG. Furthermore, we show here that FDG inhibits retrograde Shiga toxin transport and is much more efficient in protecting cells against the toxin than 2DG. In summary, our data reveal novel effects of FDG on cellular transport and glycosphingolipid metabolism, which suggest a potential clinical application of FDG as an adjuvant for cancer chemotherapy.


Subject(s)
Fluorodeoxyglucose F18/pharmacology , Lipid Metabolism/drug effects , Metabolome/drug effects , Biological Transport/drug effects , Biological Transport/radiation effects , Cells, Cultured , Endocytosis/drug effects , Endocytosis/radiation effects , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/radiation effects , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , Golgi Apparatus/radiation effects , HT29 Cells , Humans , Lipid Metabolism/radiation effects , MCF-7 Cells , Metabolome/radiation effects , Protein Transport/drug effects , Protein Transport/radiation effects , Shiga Toxin/metabolism
17.
Int J Oncol ; 49(6): 2217-2226, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27748893

ABSTRACT

Autophagy induced by radiation is critical to cell fate decision. Evidence now sheds light on the importance of autophagy induced by cancer radiotherapy. Traditional view considers radiation can directly or indirectly damage DNA which can activate DNA damage the repair signaling pathway, a large number of proteins participating in DNA damage repair signaling pathway such as p53, ATM, PARP1, FOXO3a, mTOR and SIRT1 involved in autophagy regulation. However, emerging recent evidence suggests radiation can also cause injury to extranuclear targets such as plasma membrane, mitochondria and endoplasmic reticulum (ER) and induce accumulation of ceramide, ROS, and Ca2+ concentration which activate many signaling pathways to modulate autophagy. Herein we review the role of autophagy in radiation therapy and the potent intracellular autophagic triggers induced by radiation. We aim to provide a more theoretical basis of radiation-induced autophagy, and provide novel targets for developing cytotoxic drugs to increase radiosensitivity.


Subject(s)
Autophagy/radiation effects , Endoplasmic Reticulum Stress/radiation effects , Endoplasmic Reticulum/radiation effects , Mitochondria/radiation effects , Neoplasms/radiotherapy , DNA Damage/genetics , DNA Damage/radiation effects , DNA Repair/genetics , DNA Repair/radiation effects , Endoplasmic Reticulum/pathology , Humans , Mitochondria/pathology , Neoplasms/genetics , Reactive Oxygen Species/metabolism
18.
Cell Physiol Biochem ; 39(5): 1746-1760, 2016.
Article in English | MEDLINE | ID: mdl-27744450

ABSTRACT

BACKGROUND/AIMS: Sonodynamic therapy (SDT) is considered a new approach for the treatment of atherosclerosis. We previously confirmed that hydroxyl acetylated curcumin (HAC) was a sonosensitizer. In this study, we investigated the mechanism of THP-1 macrophage apoptosis and autophagy induced by HAC mediated SDT (HAC-SDT). METHODS: Cell viability was measured using a CCK-8 assay. Laser scanning confocal microscopy was used to measure the levels of intracellular reactive oxygen species (ROS), sub-cellular HAC localization, BAX and cytochrome C translocation, LC3 expression, monodansylcadaverine staining and Dil-labeled oxidized low density lipoprotein (Dil-ox-LDL) uptake. Flow cytometry was used to analyze apoptosis and autophagy via Annexin V/propidium iodide and acridine orange staining, respectively. The expression levels of apoptosis- and autophagy-related proteins were detected by Western blot. Oil red O was used to measure intracellular lipid accumulation. RESULTS: We identified HAC (5.0 µg/mL) located in lysosomes, endoplasmic reticulum, Golgi apparatus and mitochondria after 4 h of incubation. Compared with other sonosensitizers (e.g., curcumin and emodin), HAC had a more obvious sonodynamic effect on macrophages. Furthermore, the mitochondrial-caspase pathway was confirmed to play a crucial role in the HAC-SDT-induced apoptosis; BAX translocated from the cytosol to the mitochondria during HAC-SDT. Subsequently, mitochondrial cytochrome C was released into the cytosol, activating the caspase cascade in a time-dependent manner. Furthermore, HAC-SDT could induce PI3K/AKT/mTOR pathway dependent autophagy, accompanied by a decrease in the lipid uptake of THP-1 macrophages. This mechanism was demonstrated by the formation of acidic vesicular organelles, the conversion of LC3 I to LC3 II, the expression of related proteins, and the attenuation of both Dil-ox-LDL and oil red O staining. Moreover, pre-treatment with the autophagy inhibitor 3-methyladenine enhanced the HAC-SDT-induced apoptosis. Additionally, HAC-SDT-induced autophagy and apoptosis were both blocked by ROS scavenger N-acetyl-l-cysteine. CONCLUSION: The results suggested that autophagy not only played an inhibitory role in the process of apoptosis but also could effectively attenuate lipid aggregation in THP-1 macrophages during HAC-SDT. As important intracellular mediators, the ROS generated by HAC-SDT also played a crucial role in initiating apoptosis and autophagy.


Subject(s)
Autophagy/drug effects , Curcumin/pharmacology , Endoplasmic Reticulum/drug effects , Golgi Apparatus/drug effects , Lysosomes/drug effects , Macrophages/drug effects , Mitochondria/drug effects , Acetylation , Autophagy/genetics , Autophagy/radiation effects , Cell Line , Cell Survival/drug effects , Cell Survival/radiation effects , Curcumin/analogs & derivatives , Cytochromes c/genetics , Cytochromes c/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/radiation effects , Endoplasmic Reticulum/ultrastructure , Gene Expression , Golgi Apparatus/metabolism , Golgi Apparatus/radiation effects , Golgi Apparatus/ultrastructure , Humans , Hydroxylation , Lipids/chemistry , Lipoproteins, LDL/metabolism , Lysosomes/metabolism , Lysosomes/radiation effects , Lysosomes/ultrastructure , Macrophages/cytology , Macrophages/metabolism , Macrophages/radiation effects , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mitochondria/metabolism , Mitochondria/radiation effects , Mitochondria/ultrastructure , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Reactive Oxygen Species/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Ultrasonic Waves , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism
19.
Exp Mol Pathol ; 98(3): 532-9, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25825019

ABSTRACT

BACKGROUND: Radiotherapy is an established treatment modality for early and locally advanced rectal cancer as part of short course radiotherapy and long course chemoradiotherapy. The unfolded protein response (UPR) is a cellular stress response pathway often activated in human solid tumours which has been implicated in resistance to both chemotherapy and radiotherapy. This research has investigated whether the UPR pathway is upregulated in ex-vivo samples of human colorectal cancer and characterised the interaction between radiotherapy and UPR activation in two human colorectal cancer cell lines in vitro. METHODS: In vitro UPR expression was determined in response to clinical doses of radiotherapy in both the human colorectal adenocarcinoma (HT-29) cell line and a radio-resistant clone (HT-29R) using western blotting and quantitative polymerase chain reaction. The UPR was induced using a glucose deprivation culture technique before irradiation and radiosensitivity assessed using a clonogenic assay. Ex-vivo human colorectal cancer tissue was immuno-histochemically analysed for expression of the UPR marker glucose regulated protein 78 (GRP-78). RESULTS: The UPR was strongly up regulated in ex-vivo human colorectal tumours with 36 of 50 (72.0%) specimens demonstrating moderate to strong staining for the classic UPR marker GRP-78. In vitro, therapeutic doses of radiotherapy did not induce UPR activation in either radiosensitive or radioresistant cell lines. UPR induction caused significant radiosensitisation of the radioresistant cell line (HT-29R SF2Gy=0.90 S.E.M. +/-0.08; HT-29RLG SF2Gy=0.69 S.E.M. +/-0.050). CONCLUSION: This suggests that UPR induction agents may be potentially useful response modifying agents in patients undergoing therapy for colorectal cancer.


Subject(s)
Adenocarcinoma/metabolism , Colorectal Neoplasms/metabolism , Endoplasmic Reticulum/radiation effects , Radiation Tolerance , Unfolded Protein Response , Adenocarcinoma/radiotherapy , Cell Line, Tumor , Colorectal Neoplasms/radiotherapy , Endoplasmic Reticulum/metabolism , Humans , X-Rays
20.
J Dermatol Sci ; 75(1): 24-35, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24794973

ABSTRACT

BACKGROUND: Exposure of skin to ultraviolet (UV) radiation, an environmental stressor induces number of adverse biological effects (photodamage), including cancer. The damage induced by UV-irradiation in skin cells is initiated by the photochemical generation of reactive oxygen species (ROS) and induction of endoplasmic reticulum (ER) stress and consequent activation of unfolded protein response (UPR). OBJECTIVE: To decipher cellular and molecular events responsible for UV-B mediated ER stress and UPR activation in skin cells. METHODS: The study was performed on human skin fibroblast (Hs68) and keratinocyte (HaCaT) cells exposed to UV-B radiations in lab conditions. Different parameters of UVB induced cellular and molecular changes were analyzed using Western-blotting, microscopic studies and flow cytometry. RESULTS: Our results depicted that UV-B induces an immediate ROS generation that resulted in emptying of ER Ca(2+) stores inducing ER stress and activation of PERK-peIF2α-CHOP pathway. Quenching ROS generation by anti-oxidants prevented Ca(2+) release and subsequent induction of ER stress and UPR activation. UV-B irradiation induced PERK dependent G2/M phase cell cycle arrest in Hs68 and G1/S phase cell cycle arrest in HaCaT. Also our study reflects that UV-B exposure leads to loss of mitochondrial membrane potential, activation of apoptotic cascade as evident by AnnexinV/PI staining, decreased expression of Bcl-2 and increased cleavage of PARP-1 protein. CONCLUSION: UV-B induced Ca(2+) deficit within ER lumen was mediated by immediate ROS generation. Insufficient Ca(2+) concentration within ER lumen developed ER stress leading to UPR activation. These changes were reversed by use of anti-oxidants which quench ROS.


Subject(s)
Calcium Signaling/radiation effects , Endoplasmic Reticulum Stress/radiation effects , Endoplasmic Reticulum/radiation effects , Fibroblasts/radiation effects , Keratinocytes/radiation effects , Oxidative Stress/radiation effects , Ultraviolet Rays , Unfolded Protein Response/radiation effects , Antioxidants/pharmacology , Apoptosis/radiation effects , Calcium Signaling/drug effects , Cell Cycle Checkpoints/radiation effects , Cell Line , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress/drug effects , Eukaryotic Initiation Factor-2/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Keratinocytes/drug effects , Keratinocytes/metabolism , Keratinocytes/pathology , Membrane Potential, Mitochondrial/radiation effects , Oxidative Stress/drug effects , Phosphorylation , Reactive Oxygen Species/metabolism , Transcription Factor CHOP/metabolism , Unfolded Protein Response/drug effects , eIF-2 Kinase/metabolism
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