Predictive DNA damage signaling for low­dose ionizing radiation.

Numerous studies have attempted to develop biological markers for the response to radiation for broad and straightforward application in the field of radiation. Based on a public database, the present study selected several molecules involved in the DNA damage repair response, cell cycle regulation and cytokine signaling as promising candidates for low­dose radiation­sensitive markers. The HuT 78 and IM­9 cell lines were irradiated in a concentration­dependent manner, and the expression of these molecules was analyzed using western blot analysis. Notably, the activation of ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), p53 and H2A histone family member X (H2AX) significantly increased in a concentration­dependent manner, which was also observed in human peripheral blood mononuclear cells. To determine the radioprotective effects of cinobufagin, as an ATM and CHK2 activator, an in vivo model was employed using sub­lethal and lethal doses in irradiated mice. Treatment with cinobufagin increased the number of bone marrow cells in sub­lethal irradiated mice, and slightly elongated the survival of lethally irradiated mice, although the difference was not statistically significant. Therefore, KU60019, BML­277, pifithrin­α, and nutlin­3a were evaluated for their ability to modulate radiation­induced cell death. The use of BML­277 led to a decrease in radiation­induced p­CHK2 and γH2AX levels and mitigated radiation­induced apoptosis. On the whole, the present study provides a novel approach for developing drug candidates based on the profiling of biological radiation­sensitive markers. These markers hold promise for predicting radiation exposure and assessing the associated human risk.

Blue light induced ferroptosis via STAT3/GPX4/SLC7A11/FTH1 in conjunctiva epithelium in vivo and in vitro.

The prevalence of Light-emitting diodes (LEDs) has exposed us to an excessive amount of blue light (BL) which causes various ophthalmic diseases. Previous studies have shown that conjunctiva is vulnerable to BL. In this study, we aimed to investigate the underlying mechanism of BL-induced injury in conjunctiva. We placed C57BL/6 mice and human conjunctival epithelial cell lines (HCECs) under BL (440 nm ± 15 nm, 0.2 mW/cm2) to establish a BL injury model in vivo and in vitro. Immunohistochemistry and MDA assay were used to identify lipid peroxidation (LPO) in vivo. HE staining was applied to detect morphological damage of conjunctival epithelium. DCFH-DA, C11-BODIPY 581/591, Calcein-AM, and FeRhoNox™-1 probes were performed to identify ferroptosis levels in vitro. Real-time qPCR and Western blotting techniques were employed to uncover signaling pathways of blue light-induced ferroptosis. Our findings demonstrated that BL affected tear film instability and induced conjunctival epithelium injury in vivo. Ferrostatin-1 significantly alleviated blue light-induced ferroptosis in vivo and in vitro. BL downregulates the levels of solute carrier family 7 member 11 (SLC7A11), Ferritin heavy chain (FTH1), and glutathione peroxidase (GPX4) by inhibiting the activation and translocation of the Signal transducer and activator of transcription 3 (STAT3) from inducing Fe2+ burst, ROS and LPO accumulation, ultimately resulting in ferroptosis. This study will offer new insight into BL-induced conjunctival injury and LED-induced dry eye.

Regulation of cellular responses to X-ray irradiation through the activation of lysophosphatidic acid (LPA) receptor-3 (LPA3) and LPA2 in osteosarcoma cells.

Lysophosphatidic acid (LPA) binds to its specific G protein-coupled LPA receptors (LPA1 to LPA6), resulting in the activation of various cellular functions. LPA receptor-mediated signaling facilitates tumor progression in human malignancies. In the present study, we investigated whether LPA receptor-mediated signaling contributes to cellular responses to X-ray irradiation in osteosarcoma MG-63 cells. After X-ray irradiation (2, 4 and 8 Gy), LPAR2 and LPAR3 expression levels in MG-63 cells were significantly elevated in a dose-dependent manner, but no change of LPAR1 expression level was observed. The cell growth activities of MG-63 cells irradiated with X-rays (2, 4 and 8 Gy) were reduced by LPA. Conversely, LPA3 agonist (2 S)-OMPT enhanced the cell growth activities of X-ray irradiated MG-63 cells. The cell movement of MG-63 cells exposed to X-ray irradiation (8 Gy) was inhibited by (2 S)OMPT. In cell survival assay, (2 S)-OMPT suppressed the cell survival to cisplatin (CDDP) of MG-63 cells irradiated with X-rays (8 Gy). The cell survival to CDDP of X-ray irradiated cells was elevated by LPA3 knockdown. Moreover, we evaluated the effects of LPA2 on the cell survival to CDDP of MG-63 cells exposed to X-ray irradiation (8 Gy). The cell survival to CDDP of X-ray irradiated cells was increased by LPA2 agonist GRI-977143 and reduced by LPA2 knockdown. These results suggest that LPA receptor-signaling participates in the modulation of cellular functions induced by X-ray irradiation in osteosarcoma cells.

Distinguish response of low-dose radiation with different dose-rate on gene expression of human coronary artery endothelial cells: a bioinformatic study based on transcriptomic sequencing.

PURPOSE: People are exposed to low-dose radiation in medical diagnosis, occupational, or life circumstances, but the effect of low-dose radiation on human health is still controversial. The biological effects of radiation below 100 mGy are still unproven. In this study, we observed the effects of low-dose radiation (100 mGy) on gene expression in human coronary artery endothelial cells (HCAECs) and its effect on molecular signaling. MATERIALS AND METHODS: HCAECs were exposed to 100 mGy ionizing radiation at 6 mGy/h (low-dose-rate) or 288 mGy/h (high-dose-rate). After 72 h, total RNA was extracted from sham or irradiated cells for Quant-Seq 3'mRNA-Seq, and bioinformatic analyses were performed using Metascape. Gene profiling was validated using qPCR. RESULTS: Compared to the non-irradiated control group, 100 mGy of ionizing radiation at 6 mGy/h altered the expression of 194 genes involved in signaling pathways related to heart contraction, blood circulation, and cardiac myofibril assembly differentially. However, 100 mGy at 288 mGy/h altered expression of 450 genes involved in cell cycle-related signaling pathways, including cell division, nuclear division, and mitosis differentially. Additionally, gene signatures responding to low-dose radiation, including radiation dose-specific gene profiles (HIST1H2AI, RAVER1, and POTEI) and dose-rate-specific gene profiles (MYL2 for the low-dose-rate and DHRS9 and CA14 for the high-dose-rate) were also identified. CONCLUSIONS: We demonstrated that 100 mGy low-dose radiation could alter gene expression and molecular signaling pathways at the low-dose-rate and the high-dose-rate differently. Our findings provide evidence for further research on the potential impact of low-dose radiation on cardiovascular function.

Radiation-induced Bystander Effects on Glioblastoma Tumor Cells via NMDA Receptor Signaling.

Proton therapy has been widely applied on treating inaccessible and inoperable tumors, such as tumors deep within the brain or close to the critical brain stem. Nevertheless, the damaging effect of radiation for central nervous system (CNS) tumors is difficult to be confined within the irradiated region and has led to decline of neurological function in especially children with congenital CNS tumors. Currently, the involvement of n-methyl-d-aspartate (NMDA) receptors or secretary cytokines and chemokines in proton-induced bystander effects remains unclear. To understand the modulatory effects of NMDA receptor inhibition on the survival and proliferation of glioblastoma-derived cells, mesenchymal-like U373 cells were applied along with U87 neural glioblastoma cells for single doses of proton radiation at different LET in the presence or absence of pretreatment with memantine and/or collimation. Under collimation, neuronal tumor cells that are not directly irradiated (i.e., bystander cells) encounter similar biological effects potentially through cell coupling and synaptic transmission. Furthermore, whether proton LET plays a role in the mediation of bystander effect awaits to be elucidated. From this study, synaptic transmission was found to play differential roles in the proliferation of U373 and U87 cells after exposure to collimated radiation. Also, radiation-induced cell proliferation at the late stage was more correlated with bystander cell survival than early manifested γH2AX foci, suggesting that proton-induced glutamatergic synapse may act as a more important contributor than proton-induced direct effect on DNA double-stranded breaks to the late-stage responses of glioblastoma cells.

Comparison of Three Antagonists of Hedgehog Pathway to Promote Skeletal Muscle Regeneration after High Dose Irradiation.

The current geopolitical context has brought the radiological nuclear risk to the forefront of concerns. High-dose localized radiation exposure leads to the development of a musculocutaneous radiation syndrome affecting the skin and subcutaneous muscles. Despite the implementation of a gold standard treatment based on an invasive surgical procedure coupled with autologous cell therapy, a muscular defect frequently persists. Targeting the modulation of the Hedgehog (Hh) signaling pathway appears to be a promising therapeutic approach. Activation of this pathway enhances cell survival and promotes proliferation after irradiation, while inhibition by Cyclopamine facilitates differentiation. In this study, we compared the effects of three antagonists of Hh, Cyclopamine (CA), Vismodegib (VDG) and Sonidegib (SDG) on differentiation. A stable cell line of murine myoblasts, C2C12, was exposed to X-ray radiation (5 Gy) and treated with CA, VDG or SDG. Analysis of proliferation, survival (apoptosis), morphology, myogenesis genes expression and proteins production were performed. According to the results, VDG does not have a significant impact on C2C12 cells. SDG increases the expression/production of differentiation markers to a similar extent as CA, while morphologically, SDG proves to be more effective than CA. To conclude, SDG can be used in the same way as CA but already has a marketing authorization with an indication against basal cell cancers, facilitating their use in vivo. This proof of concept demonstrates that SDG represents a promising alternative to CA to promotes differentiation of murine myoblasts. Future studies on isolated and cultured satellite cells and in vivo will test this proof of concept.

Photobiomodulation at 660 nm Stimulates In Vitro Diabetic Wound Healing via the Ras/MAPK Pathway.

Diabetic foot ulcers (DFUs) are open chronic wounds that affect diabetic patients due to hyperglycaemia. DFUs are known for their poor response to treatment and frequently require amputation, which may result in premature death. The present study evaluated the effect of photobiomodulation (PBM) at 660 nm on wound healing via activation of Ras/MAPK signalling in diabetic wounded cells in vitro. This study used four human skin fibroblast cell (WS1) models, namely normal (N), wounded (W), diabetic (D), and diabetic wounded (DW). Cells were irradiated at 660 nm with 5 J/cm2. Non-irradiated cells (0 J/cm2) served as controls. Cells were incubated for 24 and 48 h post-irradiation, and the effect of PBM on cellular morphology and migration rate, viability, and proliferation was assessed. Basic fibroblast growth factor (bFGF), its phosphorylated (activated) receptor FGFR, and phosphorylated target proteins (Ras, MEK1/2 and MAPK) were determined by enzyme-linked immunosorbent assay (ELISA) and Western blotting; nuclear translocation of p-MAPK was determined by immunofluorescence. PBM resulted in an increase in bFGF and a subsequent increase in FGFR activation. There was also an increase in downstream proteins, p-Ras, p-MEK1/2 and p-MAPK. PBM at 660 nm led to increased viability, proliferation, and migration as a result of increased bFGF and subsequent activation of the Ras/MAPK signalling pathway. Therefore, this study can conclude that PBM at 660 nm stimulates in vitro diabetic wound healing via the bFGF-activated Ras/MAPK pathway.

The ALK1­Smad1/5­ID1 pathway participates in tumour angiogenesis induced by low­dose photodynamic therapy.

Photodynamic therapy (PDT) is an effective and low­invasive tumour therapy. However, it can induce tumour angiogenesis, which is a main factor leading to tumour recurrence and metastasis. Activin receptor­like kinase­1 (ALK1) is a key factor regulating angiogenesis. However, it remains unclear whether ALK1 plays an unusual role in low­dose PDT­induced tumour angiogenesis. In the present study, human umbilical vein endothelial cells (HUVECs) co­cultured with breast cancer MDA­MB­231 cells (termed HU­231 cells) were used to construct an experimental model of tumour angiogenesis induced by low­dose PDT. The viability, and the proliferative, invasive, migratory, as well as the tube­forming ability of the HU­231 cells were evaluated following low­dose PDT. In particular, ALK1 inhibitor and and an adenovirus against ALK1 were used to further verify the role of ALK1 in low­dose PDT­induced tumour angiogenesis. Moreover, the expression of ALK1, inhibitor of DNA binding 1 (ID1), Smad 1, p­Smad1/5, AKT and PI3K were detected in order to verify the underlying mechanisms. The findings indicated that low­dose PDT enhanced the proliferative ability of the HU­231 cells and reinforced their migratory, invasive and tube formation capacity. However, these effects were reversed with the addition of an ALK1 inhibitor or by the knockdown of ALK1 using adenovirus. These results indicated that ALK1 was involved and played a critical role in tumour angiogenesis induced by low­dose PDT. Furthermore, ALK1 was found to participate in PDT­induced tumour angiogenesis by activating the Smad1/5­ID1 pathway, as opposed to the PI3K/AKT pathway. On the whole, the present study, for the first time, to the best of our knowledge, demonstrates that ALK1 is involved in PDT­induced tumour angiogenesis. The inhibition of ALK1 can suppress PDT­induced tumour angiogenesis, which can enhance the effects of PDT and may thus provide a novel treatment strategy for PDT.

Single-cell mechanistic studies of radiation-mediated bystander effects.

Ionizing radiation (IR) has been widely used in the diagnosis and treatment of clinical diseases, with radiation therapy (RT) being particularly rapid, but it can induce "bystander effects" that lead to biological responses in non-target cells after their neighboring cells have been irradiated. To help clarify how radiotherapy induces these effects, To help clarify how radiotherapy induces these effects, we analyzed single-cell RNA sequencing data from irradiated intestinal tissues on day 1 (T1 state), day 3 (T3 state), day 7 (T7 state), and day 14 (T14 state) after irradiation, as well as from healthy intestinal tissues (T0 state), to reveal the cellular level, molecular level, and involvement of different time irradiated mouse intestinal tissues in biological signaling pathways. In addition, changes in immune cell subpopulations and myeloid cell subpopulations after different radiation times were further explored, and gene regulatory networks (GRNs) of these cell subpopulations were constructed. Cellular communication between radiation-specific immune cells was explored by cell-to-cell communication events. The results suggest that radiotherapy trigger changes in immune cell subsets, which then reprogram the immune ecosystem and mediate systemic bystander effects. These radiation-specific immune cells participate in a wide range of cell-to-cell communication events. In particular, radiation-specific CD8+T cells appear to be at the core of communication and appear to persist in the body after recovery from radiotherapy, with enrichment analysis showing that radiation-specific CD8+ T cells are associated with ferroptosis. Thus, radiation-specific CD8+ T cells may be involved in cellular ferroptosis-mediated adverse effects caused by RT.

Radiation-induced Cell Death and Its Mechanisms.

ABSTRACT: With rapid technical advances, ionizing radiation has been put into wider application in ordinary living, with the worst cytological effect on the human body being cell death. Moreover, according to the Nomenclature Committee on Cell Death, the method of radiation-induced cell death, usually classified as interphase and proliferative death, undergoes more detailed classifications oriented by its molecular mechanism. Elaborating its mode and molecular mechanism is crucial for the protection and treatment of radiation injury, as well as the radiotherapy and recovery of tumors. Varying with the changes of the radiation dose and the environment, the diverse targets and pathways of ionizing radiation result in various cell deaths. This review focuses on classifications of radiation-induced cell death and its molecular mechanism. We also examine the main characteristics of ionizing radiation-induced cell death. The modes of radiation-induced cell death can be classified as apoptosis, necrosis, autophagy-dependent cell death, pyroptosis, ferroptosis, immunogenic cell death, and non-lethal processes. Once the dose is high enough, radiation effects mostly appear as destructiveness ("destructiveness" is used to describe a situation in which cells do not have the opportunity to undergo a routine death process, in which case high-dose radiation works like a physical attack). This breaks up or even shatters cells, making it difficult to find responses of the cell itself. Due to diversities concerning cell phenotypes, phases of cell cycle, radiation dose, and even cellular subregions, various methods of cell death occur, which are difficult to identify and classify. Additionally, the existence of common initial activation and signaling molecules among all kinds of cell deaths, as well as sophisticated crossways in cellular molecules, makes it more laborious to distinguish and classify various cell deaths.

Radiation-Induced Bystander Effect Mediated by Exosomes Involves the Replication Stress in Recipient Cells.

Exosomes released by irradiated cells mediate the radiation-induced bystander effect, which is manifested by DNA breaks detected in recipient cells; yet, the specific mechanism responsible for the generation of chromosome lesions remains unclear. In this study, naive FaDu head and neck cancer cells were stimulated with exosomes released by irradiated (a single 2 Gy dose) or mock-irradiated cells. Maximum accumulation of gamma H2A.X foci, a marker of DNA breaks, was detected after one hour of stimulation with exosomes from irradiated donors, the level of which was comparable to the one observed in directly irradiated cells (a weaker wave of the gamma H2A.X foci accumulation was also noted after 23 h of stimulation). Exosomes from irradiated cells, but not from control ones, activated two stress-induced protein kinases: ATM and ATR. Noteworthy is that while direct irradiation activated only ATM, both ATM and ATR were activated by two factors known to induce the replication stress: hydroxyurea and camptothecin (with subsequent phosphorylation of gamma H2A.X). One hour of stimulation with exosomes from irradiated cells suppressed DNA synthesis in recipient cells and resulted in the subsequent nuclear accumulation of RNA:DNA hybrids, which is an indicator of impaired replication. Interestingly, the abovementioned effects were observed before a substantial internalization of exosomes, which may suggest a receptor-mediated mechanism. It was observed that after one hour of stimulation with exosomes from irradiated donors, phosphorylation of several nuclear proteins, including replication factors and regulators of heterochromatin remodeling as well as components of multiple intracellular signaling pathways increased. Hence, we concluded that the bystander effect mediated by exosomes released from irradiated cells involves the replication stress in recipient cells.

Anlotinib Enhances the Antitumor Activity of High-Dose Irradiation Combined with Anti-PD-L1 by Potentiating the Tumor Immune Microenvironment in Murine Lung Cancer.

BACKGROUND: Radioimmunotherapy has become one of the most promising strategies for cancer treatment. Preclinical and clinical studies have demonstrated that antiangiogenic therapy can improve the efficacy of immunotherapy and sensitize radiotherapy through a variety of mechanisms. However, it is undefined whether angiogenesis inhibitors can enhance the effect of radioimmunotherapy. In this study, we aim to explore the role of anlotinib (AL3818) on the combination of radiotherapy and immune checkpoint inhibitors in Lewis lung carcinoma mouse. METHODS: C57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control. Immune response and immunophenotyping including the quantification and activation were determined by flow cytometry, multiplex immunofluorescence, and multiplex immunoassay. RESULTS: Triple therapy (radiotherapy combined with anti-PD-L1 and anlotinib) increased tumor-infiltrating lymphocytes and reversed the immunosuppressive effect of radiation on the tumor microenvironment in mouse model. Compared with radioimmunotherapy, the addition of anlotinib also boosted the infiltration of CD8+ T cells and M1 cells and caused a decrease in the number of MDSCs and M2 cells in mice. The levels of IFN-gamma and IL-18 were the highest in the triple therapy group, while the levels of IL-23, IL-13, IL-1 beta, IL-2, IL-6, IL-10, and Arg-1 were significantly reduced. NF-κB, MAPK, and AKT pathways were downregulated in triple therapy compared with radioimmunotherapy. Thus, the tumor immune microenvironment was significantly improved. As a consequence, triple therapy displayed greater benefit in antitumor efficacy. CONCLUSION: Our findings indicate that anlotinib might be a potential synergistic treatment for radioimmunotherapy to achieve better antitumor efficacy in NSCLC patients by potentiating the tumor immune microenvironment.

Glioblastoma recurrent cells switch between ATM and ATR pathway as an alternative strategy to survive radiation stress.

Primary treatment modality for glioblastoma (GBM) post-surgery is radiation therapy. Due to increased DNA damage repair capacity of resistant residual GBM cells, recurrence is inevitable in glioblastoma and unfortunately the recurrent tumours are resistant to the conventional therapy. Here we used our previously described in vitro radiation survival model generated from primary GBM patient samples and cell lines, which recapitulates the clinical scenario of therapy resistance and relapse. Using the parent and recurrent GBM cells from these models, we show that similar to parent GBM, the recurrent GBM cells also elicit a competent DNA damage response (DDR) post irradiation. However, the use of apical DNA damage repair sensory kinase (ATM and/or ATR) is different in the recurrent cells compared to parent cells. Consistently, we demonstrate that there is a differential clonogenic response of parent and recurrent GBM cells to the ATM and ATR kinase inhibitors with recurrent samples switching between these sensory kinases for survival emphasizing on the underlying heterogeneity within and across GBM samples. Taken together, here we report that recurrent tumours utilize an alternate DDR kinase to overcome radiation induced DNA damage. Since there is no effective treatment specifically for recurred GBM patients, these findings provide a rationale for developing newer treatment option to sensitize recurrent GBM samples by detecting in clinics the ability of cells to activate a DNA damage repair kinase different from their parent counterparts.

Activation of the atypical NF-κB pathway induced by ionizing radiation is not affected by the p53 status.

DNA double-strand breaks induced by ionizing radiation can activate the atypical NF-κB pathway via ATM-mediated phosphorylation of NEMO/IKKγ. We aimed to determine whether the status of p53 influenced the activation of this particular NF-κB pathway. The NF-κB signaling was activated either by irradiation with a single 8 Gy dose or by TNFα cytokine in p53-proficient and p53-deficient variants of HCT116, RKO, and U2-OS human cancer cell lines. To assess pathway activation the kinetics of phosphorylation (Ser32) and proteolytic degradation of IκBα inhibitor and phosphorylation (Ser536) of RelA(p65) NF-κB subunit were analyzed. Though activation of the radiation-induced atypical pathway was delayed and weakened when compared to the cytokine-induced canonical pathway, no significant differences were noted between p53-proficient and p53-deficient variants, which indicated that activation of both NF-κB pathways was not affected by the p53 status. In marked contrast, the presence of p53 significantly affected downstream effects of NF-κB activation, i.e. transcription of NF-κB-dependent genes. However, different patterns of such interference were observed, which indicated gene-specific and cell-specific mechanisms of interactions between NF-κB and p53 at the transcription regulation level.

Radiofrequency Irradiation Mitigated UV-B-Induced Skin Pigmentation by Increasing Lymphangiogenesis.

Dermal macrophages containing melanin increase skin pigmentation since dermal melanin removal is slower than epidermal melanin removal. Lymphatic vessels are also involved in melanin clearance. We evaluated whether radiofrequency (RF) irradiation induced an increase in HSP90, which promotes lymphangiogenesis by activating the BRAF/MEK/ERK pathway and decreasing tyrosinase activity, in the UV-B exposed animal model. The HSP90/BRAF/MEK/ERK pathway was upregulated by RF. Tyrosinase activity and the VEGF-C/VEGFR 3/PI3K/pAKT1/2/pERK1/2 pathway, which increase lymphangiogenesis, as well as the expression of the lymphatic endothelial marker LYVE-1, were increased by RF. Additionally, the number of melanin-containing dermal macrophages, the melanin content in the lymph nodes, and melanin deposition in the skin were decreased by RF. In conclusion, RF increased HSP90/BRAF/MEK/ERK expression, which decreased tyrosinase activity and increased lymphangiogenesis to eventually promote the clearance of dermal melanin-containing macrophages, thereby decreasing skin pigmentation.

Adaptive and Pathological Outcomes of Radiation Stress-Induced Redox Signaling.

Significance: Ionizing radiation can damage cells either directly or through oxidative damage caused by ionization. Although radiation exposure from natural sources is very limited, ionizing radiation in nuclear disaster zones and long spaceflights causes inconspicuous, yet measurable physiological effects in men and animals, whose significance remains poorly known. Understanding the physiological impacts of ionizing radiation has a wide importance due to the increased use of medical imaging and radiotherapy. Recent Advances: Radiation exposure has been traditionally investigated from the perspective of DNA damage and its consequences. However, recent studies from Chernobyl as well as spaceflights have provided interesting insights into oxidative stress-induced metabolic alterations and disturbances in the circadian regulation. Critical Issues: In this review, we discuss the physiological consequences of radiation exposure in the light of oxidative stress signaling. Radiation exposure likely triggers many converging or interconnecting signaling pathways, some of which mimic mitochondrial dysfunction and might explain the observed metabolic changes. Future Directions: Better understanding of the different radiation-induced signaling pathways might help to devise strategies for mitigation of the long-term effects of radiation exposure. The utility of fibroblast growth factor 21 (FGF21) as a radiation exposure biomarker and the use of radiation hormesis as a method to protect astronauts on a prolonged spaceflight, such as a mission to Mars, should be investigated. Antioxid. Redox Signal. 37, 336-348.

Epigallocatechin gallate enhances human lens epithelial cell survival after UVB irradiation via the mitochondrial signaling pathway.

The aim of the present study was to explore the mechanism underlying the ultraviolet B (UVB) irradiation­induced apoptosis of human lens epithelial cells (HLECs), and to investigate the protective effect of epigallocatechin gallate (EGCG) against the UVB­induced apoptosis of HLECs. HLECs were exposed to different concentrations of EGCG plus UVB (30 mJ/cm2). Cell viability was determined using the MTT assay. Furthermore, mitochondrial membrane potential (Δψm) and apoptosis were assessed by flow cytometry with JC­1 and Annexin V/PI staining, respectively. Moreover, the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH­Px), as well as the levels of GSH, hydrogen peroxide (H2O2) and hydroxyl free radicals were determined using biochemical assay techniques. Reverse transcription­quantitative PCR and western blotting were used to detect the mRNA and protein expression levels of Bcl­2, Bax, cytochrome c, caspase­9 and caspase­3, respectively. The results revealed that UVB irradiation reduced the Δψm of HLECs and induced apoptosis. Notably, EGCG significantly attenuated the generation of H2O2 and hydroxyl free radicals caused by UVB irradiation in HLECs, and significantly increased CAT, SOD and GSH­Px activities, however, the GSH levels were not significantly increased. EGCG also reduced UVB­stimulated Bax, cytochrome c, caspase­9 and caspase­3 expression, and elevated Bcl­2 expression, suggesting that EGCG may possess free radical­scavenging properties, thus increasing cell viability. In conclusion, EGCG may be able to protect against UVB­induced HLECs apoptosis through the mitochondria­mediated apoptotic signaling pathway, indicating its potential application in clinical practice.

Modulation of mTOR signaling by radiation and rapamycin treatment in canine mast cell cancer cells.

Rapamycin has been reported to reduce cancer cell survival in certain tumors following radiation therapy, but the mechanisms driving this phenomenon are unclear. Rapamycin inhibits mTOR signaling, a pathway responsible for several essential cell functions. The objective of this study was to investigate the effects of rapamycin and radiation on the activation and inhibition of mTOR signaling and the relationship between mTOR signaling and DNA damage response in vitro using canine mast cell tumor (MCT) cancer cell lines. Rapamycin rapidly inhibited S6K phosphorylation in a dose-dependent manner. Ionizing radiation (3, 6, or 10 Gy) was able to activate mTOR signalling, but the combination of radiation and rapamycin maintained mTOR inhibition. The comet assay revealed that co-treatment with rapamycin induced modest increases in the severity of DNA damage to MCT cells, but that these differences were not statistically significant. Although the relationship between mTOR and DNA damage response in MCT cancer cell lines remains unclear, our findings suggest the possibility of interaction, leading to enhancement of radiation response.

Il a été rapporté que la rapamycine réduisait la survie des cellules cancéreuses dans certaines tumeurs après une radiothérapie, mais les mécanismes à l'origine de ce phénomène ne sont pas clairs. La rapamycine inhibe la signalisation mTOR, une voie responsable de plusieurs fonctions cellulaires essentielles. L'objectif de cette étude était d'étudier les effets de la rapamycine et des radiations sur l'activation et l'inhibition de la signalisation mTOR et la relation entre la signalisation mTOR et la réponse aux dommages à l'ADN in vitro à l'aide de lignées cellulaires cancéreuses de tumeurs mastocytaires canines (MCT). La rapamycine a rapidement inhibé la phosphorylation de S6K de manière dose-dépendante. Le rayonnement ionisant (3, 6 ou 10 Gy) a pu activer la signalisation mTOR, mais la combinaison de rayonnement et de rapamycine a maintenu l'inhibition de mTOR. Le test des comètes a révélé que le co-traitement avec la rapamycine induisait des augmentations modestes de la gravité des dommages à l'ADN des cellules MCT, mais que ces différences n'étaient pas statistiquement significatives. Bien que la relation entre mTOR et la réponse aux dommages à l'ADN dans les lignées cellulaires cancéreuses MCT reste incertaine, nos résultats suggèrent la possibilité d'une interaction, conduisant à une amélioration de la réponse aux radiations.(Traduit par Docteur Serge Messier).

Regulation of the Receptor Tyrosine Kinase AXL in Response to Therapy and Its Role in Therapy Resistance in Glioblastoma.

The receptor tyrosine kinase AXL (RTK-AXL) is implicated in therapy resistance and tumor progression in glioblastoma multiforme (GBM). Here, we investigated therapy-induced receptor modifications and how endogenous RTK-AXL expression and RTK-AXL inhibition contribute to therapy resistance in GBM. GBM cell lines U118MG and SF126 were exposed to temozolomide (TMZ) and radiation (RTX). Receptor modifications in response to therapy were investigated on protein and mRNA levels. TMZ-resistant and RTK-AXL overexpressing cell lines were exposed to increasing doses of TMZ and RTX, with and without RTK-AXL tyrosine kinase inhibitor (TKI). Colorimetric microtiter (MTT) assay and colony formation assay (CFA) were used to assess cell viability. Results showed that the RTK-AXL shedding product, C-terminal AXL (CT-AXL), rises in response to repeated TMZ doses and under hypoxia, acts as a surrogate marker for radio-resistance. Endogenous RTX-AXL overexpression leads to therapy resistance, whereas combination therapy of TZM and RTX with TKI R428 significantly increases therapeutic effects. This data proves the role of RTK-AXL in acquired and intrinsic therapy resistance. By demonstrating that therapy resistance may be overcome by combining AXL TKI with standard treatments, we have provided a rationale for future study designs investigating AXL TKIs in GBM.

Photobiomodulation enhances facial nerve regeneration via activation of PI3K/Akt signaling pathway-mediated antioxidant response.

Facial nerve dysfunction is a common clinical condition that leads to disfigurement and emotional distress in the affected individuals. This study aimed to evaluate whether photobiomodulation can enhance regeneration of crushed facial nerves and attempt to investigate the possible underlying mechanism of neuroprotective function and therapeutic target. Various parameters of photobiomodulation were assigned to the facial nerves and Schwann cells (SCs) separately during crushed injury in rats. Axonal regeneration, functional outcomes, and SC apoptosis, proliferation, and underlying mechanisms of action were evaluated by morphological, histopathological, and functional assessments, flow cytometry, western blotting, real-time PCR, and IncuCyte. The results showed that photobiomodulation improved axonal regeneration and functional recovery, and also promoted proliferation, and inhibited apoptosis of SCs, both of these were considered as the most effective parameters in 250mW group. In addition, the neuroprotective effects of photobiomodulation (500mW) were likely associated with oxidative stress-induced SC apoptosis via activation of the PI3K/Akt signaling pathway. Our results revealed that photobiomodulation significantly promoted axonal regeneration, functional recovery, and regeneration of the facial nucleus, and its mechanism was related to the up-regulation of the PI3K/Akt signaling pathway. These findings provide clear experimental evidence of photobiomodulation as an alternative therapeutic strategy for peripheral nerve damage.