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.

An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage.

Targeting the molecular pathways underlying the cardiotoxicity associated with thoracic irradiation and doxorubicin (Dox) could reduce the morbidity and mortality associated with these anticancer treatments. Here, we find that vascular endothelial cells (ECs) with persistent DNA damage induced by irradiation and Dox treatment exhibit a fibrotic phenotype (endothelial-mesenchymal transition, EndMT) correlating with the colocalization of L1CAM and persistent DNA damage foci. We demonstrate that treatment with the anti-L1CAM antibody Ab417 decreases L1CAM overexpression and nuclear translocation and persistent DNA damage foci. We show that in whole-heart-irradiated mice, EC-specific p53 deletion increases vascular fibrosis and the colocalization of L1CAM and DNA damage foci, while Ab417 attenuates these effects. We also demonstrate that Ab417 prevents cardiac dysfunction-related decrease in fractional shortening and prolongs survival after whole-heart irradiation or Dox treatment. We show that cardiomyopathy patient-derived cardiovascular ECs with persistent DNA damage show upregulated L1CAM and EndMT, indicating clinical applicability of Ab417. We conclude that controlling vascular DNA damage by inhibiting nuclear L1CAM translocation might effectively prevent anticancer therapy-associated cardiotoxicity.

Endothelial-to-mesenchymal transition in anticancer therapy and normal tissue damage.

Endothelial-to-mesenchymal transition (EndMT) involves the phenotypic conversion of endothelial-to-mesenchymal cells, and was first discovered in association with embryonic heart development. EndMT can regulate various processes, such as tissue fibrosis and cancer. Recent findings have shown that EndMT is related to resistance to cancer therapy, such as chemotherapy, antiangiogenic therapy, and radiation therapy. Based on the known effects of EndMT on the cardiac toxicity of anticancer therapy and tissue damage of radiation therapy, we propose that EndMT can be targeted as a strategy for overcoming tumor resistance while reducing complications, such as tissue damage. In this review, we discuss EndMT and its roles in damaging cardiac and lung tissues, as well as EndMT-related effects on tumor vasculature and resistance in anticancer therapy. Modulating EndMT in radioresistant tumors and radiation-induced tissue fibrosis can especially increase the efficacy of radiation therapy. In addition, we review the role of hypoxia and reactive oxygen species as the main stimulating factors of tissue damage due to vascular damage and EndMT. We consider drugs that may be clinically useful for regulating EndMT in various diseases. Finally, we argue the importance of EndMT as a therapeutic target in anticancer therapy for reducing tissue damage.

Stromal Cell-Derived Factor 1 Protects Brain Vascular Endothelial Cells from Radiation-Induced Brain Damage.

Stromal cell-derived factor 1 (SDF-1) and its main receptor, CXC chemokine receptor 4 (CXCR4), play a critical role in endothelial cell function regulation during cardiogenesis, angiogenesis, and reendothelialization after injury. The expression of CXCR4 and SDF-1 in brain endothelial cells decreases due to ionizing radiation treatment and aging. SDF-1 protein treatment in the senescent and radiation-damaged cells reduced several senescence phenotypes, such as decreased cell proliferation, upregulated p53 and p21 expression, and increased senescence-associated beta-galactosidase (SA-ß-gal) activity, through CXCR4-dependent signaling. By inhibiting extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription protein 3 (STAT3), we confirmed that activation of both is important in recovery by SDF-1-related mechanisms. A CXCR4 agonist, ATI2341, protected brain endothelial cells from radiation-induced damage. In irradiation-damaged tissue, ATI2341 treatment inhibited cell death in the villi of the small intestine and decreased SA-ß-gal activity in arterial tissue. An ischemic injury experiment revealed no decrease in blood flow by irradiation in ATI2341-administrated mice. ATI2341 treatment specifically affected CXCR4 action in mouse brain vessels and partially restored normal cognitive ability in irradiated mice. These results demonstrate that SDF-1 and ATI2341 may offer potential therapeutic approaches to recover tissues damaged during chemotherapy or radiotherapy, particularly by protecting vascular endothelial cells.

A novel Chk1-binding peptide that enhances genotoxic sensitivity through the cellular redistribution of nuclear Chk1.

Since checkpoint kinase 1 (Chk1) is an essential factor for cell viability following DNA damage, the inhibition of Chk1 has been a major focus of pharmaceutical development to enhance the sensitivity of tumor cells to chemo- and radiotherapy that damage DNA. However, due to the off-target effects of conventional Chk1-targeting strategies and the toxicity of Chk1 inhibitors, alternative strategies are required to target Chk1. To facilitate such efforts, in this study, we identified a specific Chk1-binding 12-mer peptide from the screening of a phage display library and characterized the peptide in terms of cellular cytotoxicity, and in terms of its effect on Chk1 activity and sensitivity to genotoxic agents. This peptide, named N-terminal Chk1-binding peptide (Chk1­NP), bound the kinase domain of Chk1. Simulation of the binding revealed that the very N-terminus of the Chk1 kinase domain is the potential peptide binding site. Of note, the polyarginine-mediated internalization of Chk1­NP redistributed nuclear Chk1 with a prominent decrease in the nucleus in the absence of DNA damage. Treatment with Chk1­NP peptide alone decreased the viability of p53-defective HeLa cells, but not that of p53-functional NCI-H460 cells under normal conditions. The treatment of HeLa or NCI-H460 cells with the peptide significantly enhanced radiation sensitivity following ionizing radiation (IR) with a greater enhancement observed in HeLa cells. Moreover, the IR-induced destabilization of Chk1 was aggravated by treatment with Chk1­NP. Therefore, the decreased nuclear localization and protein levels of Chk1 seem to be responsible for the enhanced cancer cell killing following combined treatment with IR and Chk1­NP. The approach using the specific Chk1-binding peptide may facilitate the mechanistic understanding and potential modulation of Chk1 activities and may provide a novel rationale for the development of specific Chk1-targeting agents.

XIAP-associating factor 1, a transcriptional target of BRD7, contributes to endothelial cell senescence.

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is well known as an antagonist of XIAP-mediated caspase inhibition. Although XAF1 serves as a tumor-suppressor gene, the role of XAF1 in cellular senescence remains unclear. We found that XAF1 expression was increased by genotoxic agents, such as doxorubicin and ionizing radiation in pulmonary microvascular endothelial cells, consequently leading to premature senescence. Conversely, downregulation of XAF1 in premature senescent cells partially overcame endothelial cell senescence. p53 knockdown, but not p16 knockdown, abolished senescence phenotypes caused by XAF1 induction. XAF1 expression was transcriptionally regulated by Bromodomain 7 (BRD7). XAF1 induction with interferon-gamma (IFN-γ) treatment was abrogated by BRD7 knockdown, which resulted in blocking interferon-induced senescence. In lung cancer cells, XAF1 tumor suppressor activity was decreased by BRD7 knockdown, and inhibition of tumor growth by IFN-γ did not appear in BRD7-depleted xenograft tumors. These data suggest that XAF1 is involved in BRD7-associated senescence and plays an important role in the regulation of endothelial senescence through a p53-dependent pathway. Furthermore, regulation of the BRD7/XAF1 system might contribute to tissue or organismal aging and protection against cellular transformation.

Enhancement of cellular radiation sensitivity through degradation of Chk1 by the XIAP-XAF1 complex.

X-linked inhibitor of apoptosis (XIAP) and Chk1 are potential molecular targets in radiotherapy. However, their molecular association in the regulation of radiation sensitivity has been rarely studied. Here, we show that XIAP modulates radiation sensitivity by regulating stability of Chk1 in lung cancer cells. Both Chk1 and XIAP are highly expressed in various lung cancer cells. Overexpression of XIAP increased cell survival following genotoxic treatments by preventing downregulation of Chk1. However, XIAP reversed Chk1-protective activity in the presence of XIAP-associated factor 1 (XAF1) by degrading Chk1 via ubiquitination-dependent proteasomal proteolysis. The XIAP-XAF1 complex-mediated Chk1 degradation also required CUL4A and DDB1. Chk1 or XIAP was associated with DDB1 and CUL4A. Depletion of CUL4A or DDB1 prevented the XIAP-XAF1-mediated Chk1 degradation suggesting involvement of a CUL4A/DDB1-based E3 ubiquitin ligase in the process or its collaboration with XIAP E3 ligase activity. Taken together, our findings show that XIAP plays a dual role in modulation of Chk1 stability and cell viability following IR. In the absence of XAF1, XIAP stabilizes Chk1 under IR with corresponding increase of cell viability. By contrast, when XAF1 is overexpressed, XIAP facilitates Chk1 degradation, which leads to enhancement of radiation sensitivity. This selective regulation of Chk1 stability by XIAP and XAF1 could be harnessed to devise a strategy to modulate radiation sensitivity in lung cancer cells.

Characterization of DNA damage-induced cellular senescence by ionizing radiation in endothelial cells.

PURPOSE: Inhibition of growth in mammalian cells in response to damage or stress is known as cellular senescence. Increasing evidence suggests that double-strand breaks (DSB) commonly mediate cellular senescence. Recently, radiation exposure has been reported to induce premature senescence. MATERIALS AND METHODS: We investigated whether ionizing radiation (IR) at 4 Gy induces cellular senescence with DNA damage response in human umbilical vein endothelial cells (HUVEC). To determine alterations in gene expression on IR exposure, we have developed a DNA microarray analysis system that contains genes known to be involved in replicative senescence. RESULTS: The damage by IR exposure is shown to result in a variety of senescence-like phenotypes such as changes in cell morphology, decrease in cell proliferation, increase in senescence- associated ß-galactosidase (SA-ß-gal) staining, and suppression of angiogenic activity. Moreover, the expression levels of several genes associated with cell cycle regulation are remarkably increased in IR-exposed endothelial cells. We found that IGFBP5 (insulin-like growth factor binding protein 5), PLAT (plasminogen activator), SNAI2 (snail homolog 2), JAG1 (jagged 1), SPRY4 (Sprouty homolog 4), and CD44 were upregulated, whereas CFB (complement factor B), VCAM1 (vascular cell adhesion molecule 1), AQP1 (aquaporin 1), LOXL1 (lysyl oxidase-like 1), and RBPMS (RNA-binding protein with multiple splicing) were down- regulated in both radiation-damaged and old cells. CONCLUSIONS: These results imply that the IR-induced phenotype may be enhanced by alterations in genes associated with senescence.

Interferon-gamma enhances radiation-induced cell death via downregulation of Chk1.

Interferon-gamma (IFNγ) is a cytokine with roles in immune responses as well as in tumor control. Interferon is often used in cancer treatment together with other therapies. Here we report a novel approach to enhancement of cancer cell killing by combined treatment of IFNγ with ionizing radiation. We found that IFNγ treatment alone in HeLa cells induced phosphorylation of Chk1 in a time- and dose-dependent manner, and resulted in cell arrest. Moreover IFNγ treatment was correlated with attenuation of Chk1 as the treatment shortened protein half-life of Chk1. As Chk1 is an essential cell cycle regulator for viability after DNA damage, attenuation of Chk1 by IFNγ pre-treatment in HeLa cells resulted in increased cell death following ionizing radiation about 2-folds than ionizing radiation treatment alone whereas IFNγ treatment alone had little effect on cell death. X-linked inhibitor of apoptosis-associated factor 1 (XAF1), an IFN-induced gene, seems to partly regulate IFNγ-induced Chk1 destabilization and radiation sensitivity because transient depletion of XAF1 by siRNA prevented IFNγ-induced Chk1 attenuation and partly protected cells from IFNγ-enhanced radiation cell killing. Therefore the results provide a novel rationale to combine IFNγ pretreatment and DNA-damaging anti-cancer drugs such as ionizing radiation to enhance cancer cell killing.

Identification of ELAVL4 as a modulator of radiation sensitivity in A549 non-small cell lung cancer cells.

Identification of genes that modulate radiation sensitivity provides important tools to study cellular responses to ionizing radiation. We combined DNA microarrays and viability assays to identify modulators of radiation sensitivity in A549 lung cancer cells. Up-regulated genes were selected from microarray experiments and RNA expression levels were confirmed by real-time RT-PCR analysis. Cell viability assays such as clonogenic assay, MTT and FACS analysis of cell death, identified the ELAVL4 gene as a novel modulator of radiation sensitivity. ELAVL4 expression was induced following ionizing irradiation. Depletion of the ELAVL4 gene increased radiation sensitivity of A549 cells as shown by decreased surviving cell fraction following irradiation in clonogenic assay. Enhanced radiation sensitivity of ELAVL4-depleted cells was attributable to decreased cell proliferation as well as increased apoptotic cell death following irradiation. Thus the endogenous function of ELAVL4 in relation to radiation sensitivity might be the regulation of cell proliferation and death. This approach to identification of modulators for radiation sensitivity has several advantages in terms of functional selectivity, stringency and time. Further analysis of the modulators should find potential use in the application of radiation biomarkers as well as modulators of cellular radiation responses.

Enhancement of radiation sensitivity in lung cancer cells by celastrol is mediated by inhibition of Hsp90.

The radiosensitizing activity of celastrol, a quinone methide triterpene was examined. We found that celastrol treatment of the NCI-H460 lung cancer cell line increased radiation-induced cell killing. The increased radiosensitivity was correlated with decreased levels of Hsp90 clients, such as EGFR, ErbB2 and survivin as well as with increased p53 expression. Celastrol inhibited the ATP-binding activity of Hsp90. Furthermore, celastrol treatment dissociated an Hsp90 client protein, EGFR, and this in turn resulted in degradation of the client protein. These results were not observed with another structurally similar triterpenoid, 6ß-acetonyl-22ß-hydroxytingenol (TG), suggesting that a specific structural feature of the triterpenoid is required for radiosensitization. Moreover celastrol treatment increased p53 levels by phosphorylating Ser15 and Ser20 residues as well as by inhibiting its proteasomal degradation. Celastrol may be considered an effective radiosensitizer acting as an inhibitor of Hsp90 and a p53 activator. The two activities could be applicable to a broad range of cancer cells with either wild-type or mutant p53 because either activity could be effective for the enhancement of radiation cell killing. Further analysis with other triterpenoids should identify the functional moiety of the structure and additional candidates for effective radiosensitizers, which can be used in combined radiotherapy.

Proto-oncogenic H-Ras, K-Ras, and N-Ras are involved in muscle differentiation via phosphatidylinositol 3-kinase.

Oncogenic H-Ras G12V and its variants have been shown to inhibit muscle differentiation. However, the role of proto-oncogenic Ras (c-Ras) in muscle differentiation remains unclear. The active GTP-bound form of Ras has been known to associate with diverse effectors including Raf, phosphatidylinositol 3-kinase (PI3K), Ral-GDS, and other molecules to transmit downstream signals. We hypothesize that c-Ras may stimulate muscle differentiation by selectively activating PI3K, an important mediator for muscle differentiation. In our experiments, inhibition of c-Ras by farnesyltransferase inhibitors and a dominant negative form of H-Ras (Ras S17N) suppressed muscle differentiation. Consistently, individual knockdown of H-Ras, K-Ras, and N-Ras by siRNAs all blocked muscle differentiation. Interestingly, we found that c-Ras preferentially interacts with PI3K rather than its major binding partner c-Raf, during myogenic differentiation, with total c-Ras activity remaining unchanged. PI3K and its downstream myogenic pathway, the Nox2/NF-kappaB/inducible nitric oxide synthase (iNOS) pathway, were found to be suppressed by inhibition of c-Ras activity during differentiation. Furthermore, expression of a constitutively active form of PI3K completely rescued the differentiation block and reactivated the Nox2/NF-kappaB/iNOS pathway in c-Ras-inhibited cells. On the basis of our results, we conclude that contrary to oncogenic Ras, proto-oncogenic H-Ras, K-Ras, and N-Ras are directly involved in the promotion of muscle differentiation via PI3K and its downstream signaling pathways. In addition, PI3K pathway activation is associated with a concurrent suppression of the otherwise predominantly activated Raf/Mek/Erk pathway.

Endogenous hydrogen peroxide regulates glutathione redox via nuclear factor erythroid 2-related factor 2 downstream of phosphatidylinositol 3-kinase during muscle differentiation.

We reported previously that endogenous reactive oxygen species (ROS) function as myogenic signaling molecules. It has also been determined that excess ROS induce electrophile-response element (EpRE)-driven gene expression via activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Nonetheless, the relationship between the metabolism of ROS (eg, H(2)O(2)) through glutathione (GSH) up-regulation, GSH-dependent reduction of H(2)O(2), and Nrf2-dependent gene regulation is not well established. Therefore, we attempted to determine whether H(2)O(2) controls the intracellular GSH redox state via the Nrf2-glutamate-cysteine ligase (GCL)/glutathione reductase (GR)-GSH signaling pathway. In our experiments, enhanced H(2)O(2) generation was accompanied by an increase in both total GSH levels and the GSH/GSSG ratio during muscle differentiation. Both GCL and GR transcriptional expression levels were markedly increased during muscle differentiation but reduced by catalase treatment. Nrf2 protein expression and nuclear translocation increased during myogenesis. The inhibition of GCL, GR, and Nrf2 both by inhibitors and by RNA interference blocked muscle differentiation. Phosphatidylinositol 3-kinase regulated the expression of the GCL C (a catalytic subunit) and GR genes via the induction of Nrf2 nuclear translocation and expression. In conclusion, endogenous H(2)O(2) generated during muscle differentiation not only functions as a signaling molecule, but also regulates the GSH redox state via activation of the Nrf2-GCL/GR-GSH signaling pathway downstream of phosphatidylinositol 3-kinase.

Suppression of c-Src activity stimulates muscle differentiation via p38 MAPK activation.

Role of c-Src in muscle differentiation has been controversial. Here, we investigated if c-Src positively or negatively regulates muscle differentiation, using H9c2 and C2C12 cell lines. Inhibition of c-Src by treatment with PP1 and SU6656, pharmacologic inhibitors of Src family kinases, or by expression of a dominant negative c-Src, all induced muscle differentiation in proliferation medium (PM). In differentiating cells in differentiation medium (DM), c-Src activity gradually decreased and reached basal level 3 days after induction of differentiation. Inhibition of c-Src suppressed Raf/MEK/ERK pathway but activated p38 MAPK. Inhibition of p38 MAPK did not affect c-Src activity in PM. However, it reactivated Raf/MEK/ERK pathway in c-Src-inhibited cells regardless of PM or DM. Concomitant inhibition of c-Src and p38 MAPK activities blocked muscle differentiation in both media. In conclusion, suppression of c-Src activity stimulates muscle differentiation by activating p38 MAPK uni-directionally.

Overexpressed cyclophilin A in cancer cells renders resistance to hypoxia- and cisplatin-induced cell death.

Cyclophilin A (CypA) has been reported to be overexpressed in cancer cells, especially in solid tumors. To determine the role of CypA in tumorigenesis, we investigated the induction of CypA as well as the role it plays in cancer cells. Here, we have shown that induction of CypA is associated with hypoxia in a variety of cells, including DU145 human prostate cancer cell line. Our analysis of the CypA promoter clearly showed that CypA up-regulation is mediated by hypoxia-inducible factor-1alpha transcription factor. Interestingly, overexpression of CypA prevented hypoxia- and cisplatin-induced apoptosis, and this was associated with the suppression of reactive oxygen species generation and depolarization of mitochondrial membrane potential, whereas small interfering RNA-based CypA knockdown aggravated these factors. These results suggest that CypA is important in tumorigenesis, especially in tumor apoptosis.

Prednisolone suppresses cyclosporin A-induced apoptosis but not cell cycle arrest in MDCK cells.

Cyclosporin A (CsA) is a potent immunosuppressive agent, and can cause severe adverse effects including nephrotoxicity partly due to generation of reactive oxygen species (ROS). Glucocorticoids, which are widely used in combination with CsA, have been shown to reduce oxidative injuries in various cells, but its mechanism is not understood well. To investigate the effects of prednisolone (Pd) on CsA-induced cellular damage and ROS generation in Madin-Darby canine kidney (MDCK) tubular epithelial cells, cells were treated with CsA, CsA plus Pd, or CsA plus vitamin E. Pretreatment with Pd protected cells from CsA-induced apoptosis but not from G(0)/G(1) cell cycle arrest even at its maximal protective concentration (30 microM), whereas vitamin E almost completely inhibited both CsA-induced apoptosis and cell cycle arrest at 1 microM concentration. In addition, Pd reduced the amount of CsA-induced ROS and showed partly restored catalase which was down-regulated by 10 microM CsA at both the mRNA and protein levels. Vitamin E completely abolished CsA-induced ROS generation and catalase attenuation at 10 microM concentration. Finally, the effects of 1 microM vitamin E on CsA-induced ROS and apoptosis as well as cell cycle arrest were similar to those of 30 microM Pd. We conclude that, in MDCK cells, Pd protects against CsA-induced cytotoxicity by suppressing ROS generation, although its protective effect is weaker than that of vitamin E.

Clinical application of fresh fibroblast allografts for the treatment of diabetic foot ulcers: a pilot study.

Diabetic foot ulcers often pose a difficult problem for health care professionals because of the defects associated with fibroblast functioning. Although there has been much interest recently in the use of topical growth factors for the treatment of diabetic foot ulcers, the effects are generally not very dramatic. Cryopreserved fibroblast implants, which are able to adjust to a wound's environment and provide the desired growth factors and other substances that may be lacking in a chronic wound, represent an exciting development and a major advance. These products may well provide growth factors in the right concentration and in the right sequence, something that has proved difficult to achieve with the topical application of recombinant growth factors. However, cell activities are impaired by cryopreservation. The purpose of this study was to assess the effects of fresh human allogeneic fibroblast grafting for the treatment of diabetic foot ulcers. Eight patients with diabetic foot ulcers ranging from 6 to 17 weeks in duration were treated. The size of the wounds ranged from 2.0 to 6.0 cm2, with three patients exhibiting exposed bones. A history of diabetic foot ulcers was present in five patients. Human dermal fibroblasts from healthy teenagers were cultured in Dulbecco's modified Eagle medium/Ham's F-12 supplemented with 10% autologous serum. The cultured cells were applied over the wounds immediately after debridement; fibrin was used as a cell carrier. A dressing was then applied with Tegaderm and kept moist until healing was complete. The progress and time for complete wound closure and patient satisfaction were assessed, with follow-up time ranging from 6 to 18 months. Complete wound healing occurred in all patients. Eleven to 21 days were needed for complete reepithelization of the wound, and no clinical or laboratory abnormalities were noted. Patient satisfaction was also very positive. In this study, the use of fresh human fibroblast allografts was found to be a safe and effective treatment for diabetic foot ulcers.