KTN1 mediated unfolded protein response protects keratinocytes from ionizing radiation-induced DNA damage.

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.

Red and near-infrared light evokes Ca2+ influx, endoplasmic reticulum release and membrane depolarization in neurons and cancer cells.

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.

Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma.

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.

Role of Endoplasmic Reticulum and Mitochondrion in Proton Microbeam Radiation-Induced Bystander Effect.

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.

PERK Regulates Glioblastoma Sensitivity to ER Stress Although Promoting Radiation Resistance.

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.

Endoplasmic Reticulum-Localized Two-Photon-Absorbing Boron Dipyrromethenes as Advanced Photosensitizers for Photodynamic Therapy.

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.

Transmission electron microscopy method providing high resolution in the cell section without radiation damage.

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.

Comparison of Ca2+ puffs evoked by extracellular agonists and photoreleased IP3.

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.

Cellular effects of fluorodeoxyglucose: Global changes in the lipidome and alteration in intracellular transport.

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.

Crosstalk between autophagy and intracellular radiation response (Review).

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.

Combination of Hydroxyl Acetylated Curcumin and Ultrasound Induces Macrophage Autophagy with Anti-Apoptotic and Anti-Lipid Aggregation Effects.

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.

Targeting the endoplasmic reticulum mediates radiation sensitivity in colorectal cancer.

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.

Oxidative stress mediated Ca(2+) release manifests endoplasmic reticulum stress leading to unfolded protein response in UV-B irradiated human skin cells.

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.

Endoplasmic reticulum targeted chemotherapeutics: the remarkable photo-cytotoxicity of an oxovanadium(IV) vitamin-B6 complex in visible light.

An oxovanadium(IV) vitamin-B6 Schiff base complex, viz. [VO(HL)(acdppz)]Cl, having (acridinyl)dipyridophenazine (acdppz) shows specific localization to endoplasmic reticulum (ER) and remarkable apoptotic photocytotoxicity in visible light (400-700 nm) in HeLa and MCF-7 cancer cells (IC50 < 0.6 µM) while being non-toxic in the dark and to MCF-10A normal cells (IC50 > 40 µM).

Induction of endoplasmic reticulum stress by sonoporation: linkage to mitochondria-mediated apoptosis initiation.

The use of cavitational means to create transient membrane pores on living cells (i.e., sonoporation) may potentially induce a broad range of downstream bio-effects that disrupt the functioning of various organelles. Here we observed that on HL-60 leukemia cells, sonoporation may induce endoplasmic reticulum (ER) stress on a time-lapse basis and, in turn, signal the mitochondria to commit a cell toward apoptosis. Our observations were derived from in vitro ultrasound exposure experiments performed on HL-60 cells in the presence of lipid-shelled microbubbles (1:1 cell-to-bubble ratio; 1-MHz frequency; 0.45-MPa in situ peak negative pressure; 100-cycle pulse length; 1-kHz pulse repetition frequency; 60-s exposure period). Using flow cytometry, we found that sonoporated cells exhibited a progressive loss of functional ER mass over a 6-h period. Also, post-exposure Western blot assays (between 0 and 24 h) revealed various indications of post-sonoporation ER stress: (i) upregulation of ER-resident enzymes responsible for catalyzing protein folding; (ii) activation of trans-ER-membrane stress sensors; (iii) increased expression of ER-induced regulatory proteins that mediate pro-apoptotic signals to the mitochondria. These results corresponded to flow cytometry observations that depicted a progressive depolarization of a sonoporated cell's mitochondrial outer membrane potential. They were also consistent with another Western blot assay that found, in sonoporated cells, a time-lapse increase of caspase-9 (a mitochondria-activated apoptosis initiator protein). Taken together, our findings indicate that sonoporation may upset ER homeostasis, and this may ultimately result in initiation of apoptosis.

Silibinin is a potent sensitizer of UVA radiation-induced oxidative stress and apoptosis in human keratinocyte HaCaT cells.

UVA radiation (315-400 nm), which constitutes ca 95% of the UV irradiation in natural sunlight reaching earth surface, is a major environmental risk factor associated with human skin cancer pathogenesis. UVA is an oxidizing agent that causes significant damage to cellular components through the release of reactive oxygen species (ROS) and leads to photoaging and photocarcinogenesis. Here we investigate the effect of silibinin, the flavonolignan from Silybum marianum, on UVA-induced ROS and cell death in human keratinocyte cell line HaCaT. In addition, the effect of silibinin on UVA-induced intracellular ROS-mediated endoplasmic reticulum (ER) stress was also analyzed. UVA irradiation resulted in ROS production and apoptosis in HaCaT cells in a dose-dependent manner, and the ROS levels and apoptotic index were found to be elevated significantly when the cells were treated with 75 µmsilibinin for 2 h before UVA exposure. When the cells were pretreated with 10 mmN-acetyl cysteine, the enhancement of UVA-induced apoptosis by silibinin was compromised. Furthermore, we found that silibinin enhances ER stress-mediated apoptosis in HaCaT cells by increasing the expression of CHOP protein. These results suggest that silibinin may be beneficial in the removal of UVA-damaged cells and the prevention of skin cancer.

Crocetin prevents retinal degeneration induced by oxidative and endoplasmic reticulum stresses via inhibition of caspase activity.

Crocetin is a carotenoid that is the aglicone of crocin, which are found in saffron stigmas (Crocus sativus L.) and gardenia fruit (Gardenia jasminoides Ellis). In this study, we investigated the effects of crocetin on retinal damage. To examine whether crocetin affects stress pathways, we investigated intracellular oxidation induced by reactive oxygen species, expression of endoplasmic reticulum (ER) stress-related proteins, disruption of the mitochondrial membrane potential (ΔΨ(m)), and caspases activation. In vitro, we employed cultured retinal ganglion cells (RGC-5, a mouse ganglion cell-line transformed using E1A virus). Cell damage was induced by tunicamycin or hydrogen peroxide (H(2)O(2)) exposure. Crocetin at a concentration of 3µM showed the inhibitory effect of 50-60% against tunicamycin- and H(2)O(2)-induced cell death and inhibited increase in caspase-3 and -9 activity. Moreover, crocetin inhibited the enzymatic activity of caspase-9 in a cell-free system. In vivo, retinal damage in mice was induced by exposure to white light at 8000lx for 3h after dark adaptation. Photoreceptor damage was evaluated by measuring the outer nuclear layer thickness at 5days after light exposure and recording the electroretinogram (ERG). Retinal cell damage was also detected with Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining at 48h after light exposure. Crocetin at 100mg/kg, p.o. significantly inhibited photoreceptor degeneration and retinal dysfunction and halved the expression of TUNEL-positive cells. These results indicate that crocetin has protective effects against retinal damage in vitro and in vivo, suggesting that the mechanism may inhibit increase in caspase-3 and -9 activities after retinal damage.

Rose bengal acetate photodynamic therapy-induced autophagy.

Photodynamic therapy (PDT), an anticancer therapy requiring the exposure of cells or tissue to a photosensitizing drug followed by irradiation with visible light of the appropriate wavelength, induces cell death by the efficient induction of apoptotic as well as non-apoptotic mechanisms, such as necrosis and autophagy, or a combination of all three mechanisms. However, the exact role of autophagy in photodynamic therapy is still a matter of debate. To understand the role of autophagy in PDT, we investigated the induction of autophagy in HeLa cells photosensitized with Rose Bengal Acetate (RBAc). After incubation with Rose Bengal Acetate (10-5 M), HeLa cells were irradiated for 90 seconds (green LED DPL 305, emitting at 530 +15 nm to obtain 1.6 J/cm2 as the total light dose) and allowed to recover for 72 h. Induction of autophagy and apoptosis were observed with peaks at 8 h and 12 h after irradiation, respectively. Autophagy was detected by biochemical (Western Blotting for the LC3B protein) and morphological criteria (TEM, cytochemistry). In addition, the pan-caspase inhibitor, z-VAD, was unable to completely prevent cell death. The simultaneous onset of apoptosis and autophagy following Rose Bengal Acetate PDT is of remarkable interest in light of the findings that autophagy can result in the class II presentation of antigens and thus, explain why low dose PDT can yield anti-tumor immune responses.

ER stress induced by ionising radiation in IEC-6 cells.

PURPOSE: Ionising radiation (IR) can evoke a series of biochemical events inside the cell. However, whether IR can directly induce endoplasmic reticulum (ER) stress is not clear. In our previous study, we found that there might be a causative link between IR and ER stress. In this study, we further characterised the type of ER stress induced by IR. MATERIALS AND METHODS: Rat intestinal epithelial cells IEC-6 were irradiated at a dose of 10 Gy, and total RNA and proteins were harvested at indicated time points. The mRNA and protein expression of immunoglobulin heavy chain binding protein (BiP) and glucose regulated protein 94 (GRP94) was detected along with proteins associated with ER stress signal pathways. RESULTS: Our results indicated that IR induced up-regulation of ER stress marker including BiP and GRP94 at protein and mRNA levels in IEC-6 cells. Increased phosphorylation of eukaryotic translation initiation factor 2 (eIF2alpha) and induced mRNA splicing of X-box binding protein 1 (XBP1) suggested that PERK (interferon-induced double-stranded RNA-activated protein kinase (PRKR) -like endoplasmic reticulum kinase) and IRE1 (inositol requirement 1) signal transduction pathways were involved in this kind of ER stress. However, the active form of activating transcription factor 6 (ATF6) did not change significantly in irradiated cells, which suggested that the ATF6 pathway was not involved. CONCLUSIONS: Thus, we concluded that IR could induce moderate ER stress directly in IEC-6 cells.

Endoplasmic reticulum stress mediates radiation-induced autophagy by perk-eIF2alpha in caspase-3/7-deficient cells.

As apoptosis defects limit efficacy of anticancer agents, autophagy has been proposed as a novel strategy for radiotherapy enhancement. We previously showed that caspase-3/7 inhibition induces autophagy and promotes radiosensitivity in vitro and in vivo. Therefore, we further investigated the mechanism by which radiation triggers autophagy in caspase-3/7-deficient cells, and found the involvement of endoplasmic reticulum (ER) stress. The ER activates a survival pathway, the unfolded protein response, which involves ER-localized transmembrane proteins such as protein kinase-like ER kinase (PERK), inositol-requiring enzyme-1 and activating transcription factor-6. In this study, we found that PERK is essential for radiation-induced autophagy and radiosensitivity in caspase-3/7 double-knockout cells. Irradiation of these cells increased expression of phosphorylated-eIF2alpha. Similar results were seen after administration of tunicamycin (TM), a well-known ER stressor. Importantly, we found that the administration of TM with radiation in MCF-7 breast cancer cells, which are lacking functional caspase-3 and relatively resistant to many anticancer agents, enhances radiation sensitivity. Our findings reveal ER stress as a novel potential mechanism of radiation-induced autophagy in caspase-3/7-deficient cells and as a potential strategy to maximize efficiency of radiation therapy in breast cancer.