LDR-Induced miR-30a and miR-30b Target the PAI-1 Pathway to Control Adverse Effects of NSCLC Radiotherapy.

Radiotherapy has been a central part in curing non-small cell lung cancer (NSCLC). However, it is possible that not all of the tumor cells are destroyed by radiation; therefore, it is important to effectively control residual tumor cells that could become aggressive and resistant to radiotherapy. In this study, we aimed to investigate the molecular mechanism of decreased NSCLC radioresistance by low-dose radiation (LDR) pretreatment. The results indicated that miR-30a and miR-30b, which effectively inhibited plasminogen activator inhibitor-1 (PAI-1), were overexpressed by treatment of LDR to NSCLC cells. Phosphorylation of Akt and ERK, the downstream survival signals of PAI-1, was decreased by PAI-1 inhibition. Reduced cell survival and epithelial-mesenchymal transition by PAI-1 inhibition were confirmed in NSCLC cells. Moreover, in vivo orthotopic xenograft mouse models with 7C1 nanoparticles to deliver miRNAs showed that tumor growth and aggressiveness were efficiently decreased by LDR treatment followed by radiotherapy. Taken together, the present study suggested that PAI-1, whose expression is regulated by LDR, was critical for controlling surviving tumor cells after radiotherapy.

Low dose radiation regulates BRAF-induced thyroid cellular dysfunction and transformation.

BACKGROUND: The existence of differentiated thyroid cells is critical to respond radioactive iodide treatment strategy in thyroid cancer, and loss of the differentiated phenotype is a trademark of iodide-refractive thyroid disease. While high-dose therapy has been beneficial to several cancer patients, many studies have indicated this clinical benefit was limited to patients having BRAF mutation. BRAF-targeted paired box gene-8 (PAX8), a thyroid-specific transcription factor, generally dysregulated in BRAF-mutated thyroid cancer. METHODS: In this study, thyroid iodine-metabolizing gene levels were detected in BRAF-transformed thyroid cells after low and high dose of ionizing radiation. Also, an mRNA-targeted approach was used to figure out the underlying mechanism of low (0.01Gyx10 or 0.1Gy) and high (2Gy) radiation function on thyroid cancer cells after BRAFV600E mutation. RESULTS: Low dose radiation (LDR)-induced PAX8 upregulation restores not only BRAF-suppressive sodium/iodide symporter (NIS) expression, one of the major protein necessary for iodine uptake in healthy thyroid, on plasma membrane but also regulate other thyroid metabolizing genes levels. Importantly, LDR-induced PAX8 results in decreased cellular transformation in BRAF-mutated thyroid cells. CONCLUSION: The present findings provide evidence that LDR-induced PAX8 acts as an important regulator for suppression of thyroid carcinogenesis through novel STAT3/miR-330-5p pathway in thyroid cancers.

Effects of low-dose irradiation on mice with Escherichia coli-induced sepsis.

Although favorable immune responses to low-dose irradiation (LDI) have been observed in normal mice, i.e., a hormesis effect, little is known about the effects of LDI in infectious diseases. In this study, we examined the effects of LDI on mice with sepsis, a severe and often lethal hyperinflammatory response to bacteria. Female C57BL/6 mice were whole-body irradiated with 10cGy 48h before Escherichia coli infection, and survival, bacterial clearance, cytokines, and antioxidants were quantified. LDI pretreatment significantly increased survival from 46.7% in control mice to 75% in mice with sepsis. The bacterial burden was significantly lower in the blood, spleen, and kidney of LDI-treated mice than in those of control septic mice. The levels of pro-inflammatory cytokines, e.g., IL-1ß and IL-6, as well as anti-inflammatory IL-10 were markedly reduced in pre-LDI septic mice. Nitric oxide production by peritoneal macrophages was also reduced in pre-LDI septic mice. Immune cells in the spleen increased and Nrf2 and HO-1 were induced in pre-LDI septic mice. LDI stimulates the immune response and minimizes lethality in septic mice via enhanced bacterial clearance and reduced initial proinflammatory responses.

Low-dose γ-radiation inhibits IL-1ß-induced dedifferentiation and inflammation of articular chondrocytes via blockage of catenin signaling.

Although low-dose radiation (LDR) regulates a wide range of biological processes, limited information is available on the effects of LDR on the chondrocyte phenotype. Here, we found that LDR, at doses of 0.5-2 centiGray (cGy), inhibited interleukin (IL)-1ß-induced chondrocyte destruction without causing side effects, such as cell death and senescence. IL-1ß treatment induced an increase in the expression of α-, ß-, and γ-catenin proteins in chondrocytes via Akt signaling, thereby promoting dedifferentiation through catenin-dependent suppression of Sox-9 transcription factor expression and induction of inflammation through activation of the NF-κB pathway. Notably, LDR blocked cartilage disorders by inhibiting IL-1ß-induced catenin signaling and subsequent catenin-dependent suppression of the Sox-9 pathway and activation of the NF-κB pathway, without directly altering catenin expression. LDR also inhibited chondrocyte destruction through the catenin pathway induced by epidermal growth factor, phorbol 12-myristate 13-acetate, and retinoic acid. Collectively, these results identify the molecular mechanisms by which LDR suppresses pathophysiological processes and establish LDR as a potentially valuable therapeutic tool for patients with cytokine- or soluble factors-mediated cartilage disorders.

Preventive and therapeutic effects of low-dose whole-body irradiation on collagen-induced rheumatoid arthritis in mice.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by progressive joint inflammation, resulting in cartilage destruction and bone erosion. It was reported that low-dose radiation modulates immune disease. Here, we investigated whether low-dose whole-body irradiation has preventive and therapeutic effects in collagen-induced RA (CIA) mouse models. Fractionated low-dose irradiation (0.05 Gy/fraction, total doses of 0.1, 0.5 or 0.8 Gy) was administered either concurrently with CIA induction by Type II collagen immunization (preventive) or after CIA development (therapeutic). The severity of CIA was monitored using two clinical parameters, paw swelling and redness. We also measured total Immunoglobulin G (IgG) and inflammatory cytokines (interleukine (IL)-6, IL-1ß and tumor necrosis factor-alpha (TNF-α)) in the serum by enzyme-linked immunosorbent assay, and we evaluated histological changes in the ankle joints by immunohistochemistry and hematoxylin and eosin staining. Low-dose irradiation reduced CIA clinical scores by up to 41% in the preventive model and by 28% in the therapeutic model, while irradiation in the preventive model reduced the typical CIA incidence rate from 82 to 56%. In addition, low-dose irradiation in the preventive model decreased total IgG by up to 23% and decreased IL-1ß and TNF-α by 69 and 67%, and in the therapeutic model, decreased total IgG by up to 35% and decreased IL-1ß and IL-6 by 59 and 42% with statistical significance (P < 0.01, 0.05 and 0.001). Our findings demonstrate that low-dose radiation has preventive and therapeutic anti-inflammatory effects against CIA by controlling the immune response, suggesting that low-dose radiation may represent an alternative therapy for RA, a chronic degenerative immune disease.

The effects of low-dose ionizing radiation in the activated rat basophilic leukemia (RBL-2H3) mast cells.

Mast cells play important roles in many biological responses, such as those during allergic diseases and inflammatory disorders. Although laser and UV irradiation have immunosuppressive effects on inflammatory diseases by suppressing mast cells, little is known about the effects of γ-ionizing radiation on mast cells. In this study, we investigated the effects of γ-ionizing radiation on RBL-2H3 cells, a convenient model system for studying regulated secretion by mast cells. Low-dose radiation (<0.1 gray (Gy)) did not induce cell death, but high-dose radiation (>0.5 Gy) induced apoptosis. Low-dose ionizing radiation significantly suppressed the release of mediators (histamine, ß-hexosaminidase, IL-4, and tumor necrosis factor-α) from immunoglobulin E (IgE)-sensitized RBL-2H3 cells. To determine the mechanism of mediator release inhibition by ionizing radiation, we examined the activation of intracellular signaling molecules such as Lyn, Syk, phospholipase Cγ, PKCs, and MAPK, and intracellular free calcium concentrations ([Ca(2+)](i)). The phosphorylation of signaling molecules following stimulation of high-affinity IgE receptor I (FcεRI) was specifically inhibited by low-dose ionizing radiation (0.01 Gy). These results were due to the suppression of FcεRI expression by the low-dose ionizing radiation. Therefore, low-dose ionizing radiation (0.01 Gy) may function as a novel inhibitor of mast cell activation.

A synergistic interaction between transcription factors nuclear factor-κB and signal transducers and activators of transcription 3 promotes gastric cancer cell migration and invasion.

BACKGROUND: The transcription factor nuclear factor-κB (NF-κB) has been implicated in gastric cancer metastasis, but the underlying molecular mechanisms remain unclear. We investigated the role of the interaction between NF-κB and signal transducers and activators of transcription 3 (STAT3) in controlling metastatic potential of gastric cancer cells. METHODS: Immunohistochemistry for NF-κB p65 (RelA), phospho-Tyr705-STAT3 (pSTAT3), or matrix metalloproteinase 9 (MMP9) was performed on tissue array slides containing 255 gastric carcinoma specimens. NF-κB inhibition in SNU-638 and MKN1 gastric cancer cell lines were performed by transduction with a retroviral vector containing NF-κB repressor mutant of IκBα, and STAT3 was silenced by RNA interference. We also did luciferase reporter assay, double immunofluorescence staining and immunoblotting. Cell migration and invasion were determined by wound-healing assay and invasion assay, respectively. RESULTS: NF-κB and STAT3 were constitutively activated and were positively correlated (P=0.038) in gastric cancer tissue specimens. In cell culture experiments, NF-κB inhibition reduced STAT3 expression and activation, whereas STAT3 silencing did not affect NF-κB activation. Moreover, both NF-κB inhibition and STAT3 silencing decreased gastric cancer cell migration and invasion in a synergistic manner. In addition, both NF-κB activation and STAT3 activation were positively correlated with MMP9 in gastric cancer tissues (P=0.001 and P=0.022, respectively), decreased E-cadherin expression and increased Snail and MMP9 expressions in cultured cells. CONCLUSION: NF-κB and STAT3 are positively associated and synergistically contribute to the metastatic potential of gastric cancer cells. Thus, dual use of NF-κB and STAT3 inhibitors may enhance the efficacy of the anti-metastatic treatment of gastric cancer.

TOPORS modulates H2AX discriminating genotoxic stresses.

H2AX plays an important role in chromatin reorganization implicated in DNA repair and apoptosis under various DNA damaging conditions. In this study, the interaction between TOPORS (topoisomerase I-binding protein) and H2AX was verified using mammalian cell extracts exposed to diverse DNA damaging stresses such as ionizing radiation, doxorubicin, camptothecin, and hydrogen peroxide. In vitro assays for ubiquitination revealed that TOPORS functions as a novel E3 ligase for H2AX ubiquitination. TOPORS was found to be dissociated from H2AX proteins when cells were exposed to oxidative stress, but not replication-inducing DNA damaging stress. The protein stability of H2AX was decreased when TOPORS was ectopically expressed in cells, and oxidative stresses such as hydrogen peroxide and ionizing radiation induced recovery of the H2AX protein level. Therefore, these biochemical data suggest that TOPORS plays a key role in the turnover of H2AX protein, discriminating the type of DNA damaging stress.

Differential expression of immune-associated cancer regulatory genes in low- versus high-dose-rate irradiated AKR/J mice.

AKR/J mice carrying leukemia viral inserts develop thymic lymphoma. Recently, we demonstrated that the incidence of thymic lymphoma was decreased when these mice were raised in a low-dose-rate γ-irradiation facility. In contrast, mice irradiated at a high-dose rate developed severe thymic lymphoma and died much earlier. To understand the genetic changes occurred by low- versus high-dose-rate γ-irradiation whole genome microarray was performed. Both groups of mice demonstrated up-regulation of Ifng, Igbp1, and IL7 in their thymuses, however, mice exposed to high-dose-rate γ-irradiation exhibited marked down-regulation of Sp3, Il15, Traf6, IL2ra, Pik3r1, and Hells. In contrast, low-dose-rate irradiated mice demonstrated up-regulation of Il15 and Jag2. These gene expression profiles imply the impaired immune signaling pathways by high-dose-rate γ-irradiation while the facilitation of anti-tumor immune responses by low-dose-rate γ-irradiation. Therefore, our data delineate common and distinct immune-associated pathways downstream of low- versus high-dose-rate irradiation in the process of cancer progression in AKR/J mice.

Phosphorylation of CLK2 at serine 34 and threonine 127 by AKT controls cell survival after ionizing radiation.

AKT phosphorylates components of the intrinsic cell survival machinery and promotes survival to various stimuli. In the present study, we identified CDC-like kinase 2 (CLK2) as a new substrate of AKT activation and elucidated its role in cell survival to ionizing radiation. AKT directly binds to and phosphorylates CLK2 on serine 34 and threonine 127, in vitro and in vivo. CLK2 phosphorylation was detected in HeLa cells overexpressing active AKT. In addition, we demonstrated that ionizing radiation induces CLK2 phosphorylation via AKT activation. In contrast, the suppression of endogenous AKT expression by siRNA inhibited CLK2 phosphorylation in response to 2 gray of γ-ray or insulin. Furthermore, we examined the effect of CLK2 on the survival of irradiated CCD-18Lu cells overexpressing Myc-CLK2. CLK2 overexpression significantly increased cell growth and inhibited cell death induced by 2 gray. The role of CLK2 in cell survival to ionizing radiation was dependent on the phosphorylation of serine 34 and threonine 127. Our results suggest that AKT activation controls cell survival to ionizing radiation by phosphorylating CLK2, revealing an important regulatory mechanism required for promoting cell survival.

Genome-wide analysis of low-dose irradiated male Drosophila melanogaster with extended longevity.

Ionizing radiation generates oxidative stress, which is thought to be a major cause of aging. Although living organisms are constantly exposed to low levels of radiation, most studies examining the effect of radiation have focused on accelerated aging and diminished life span that result from high-dose radiation. On the other hand, several studies have suggested that low-dose radiation enhances the longevity of Drosophila melanogaster. Therefore, investigation of the biological effects of low-dose radiation could contribute to a more comprehensive understanding of the aging process. In this study, microarray and quantitative real time-PCR were used to measure genome-wide changes in transcript levels in low-dose irradiated fruit flies that showed enhanced longevity. In response to radiation, approximately 13% of the genome exhibited changes in gene expression, and a number of aging-related genes were significantly regulated. These data were compared with quantitative trait loci affecting life-span to identify candidate genes involved in enhanced longevity induced by low-dose radiation. This genome-wide survey revealed novel information about changes in transcript levels in low-dose irradiated flies and identified 39 new candidate genes for molecular markers of extended longevity induced by ionizing radiation. In addition, this study also suggests a mechanism by which low-dose radiation extends longevity.

Impairment of nuclear factor-kappaB activation increased glutamate excitotoxicity in a motoneuron-neuroblastoma hybrid cell line expressing mutant (G93A) Cu/Zn-superoxide dismutase.

Mutations in the superoxide dismutase 1 (SOD1) gene are linked to glutamate excitotoxicity in familial amyotrophic lateral sclerosis (fALS), but the underlying mechanism remains unclear. We investigated whether nuclear factor-kappaB (NF-kappaB) activation is involved in glutamate excitotoxicity by using motor neuron-neuroblastoma hybrid cells that expressed a mutant (G93A) SOD1 (mtSOD1) or wild-type SOD1 (wtSOD1). MtSOD1 cells were more vulnerable to glutamate excitotoxicity than wtSOD1 cells and showed higher NF-kappaB activity, higher nuclear cRel expression, and lower nuclear RelA expression under basal conditions. Glutamate treatment increased NF-kappaB activation along with nuclear expressions of RelA and cRel in wtSOD1 cells but induced only weak nuclear RelA expression in mtSOD1 cells. Suppression of NF-kappaB activation using transfection of the superrepressive mutant form of IkappaBalpha (mIkappaBalpha) inhibited nuclear RelA expression in both types of SOD1 cells, which increased glutamate excitotoxicity in wtSOD1 cells but not in mtSOD1 cells. Furthermore, immunohistochemistry confirmed stronger RelA immunoreactivity in the nuclei of motor neurons of spinal cord in wild-type SOD1 transgenic mice than in those in SOD1 G93A transgenic mice. In addition, we found that glutamate treatment decreased XIAP expression and increased caspase-3 activity in mtSOD1 cells and mIkappaBalpha-overexpressing wtSOD1 cells. Our results suggest that glutamate excitotoxicity in motor neurons of SOD1-linked fALS is attributable, at least in part, to the impairment of IkappaBalpha-dependent RelA activation and subsequent apoptosis mediated by XIAP inhibition and caspase-3 activation.

Evaluation of Anti-Tumor Effects of Whole-Body Low-Dose Irradiation in Metastatic Mouse Models.

Low-dose irradiation (LDI) has recently been shown to have various beneficial effects on human health, such as on cellular metabolic activities, DNA repair, antioxidant activity, homeostasis potency, and immune activation. Although studies on the immunogenic effects of LDI are rapidly accumulating, clinical trials for cancer treatment are considered premature owing to the lack of available preclinical results and protocols. Here, we aim to investigate anti-tumor and anti-metastatic effects of whole-body LDI in several tumor-bearing mouse models. Mice were exposed to single or fractionated whole-body LDI prior to tumor transplantation, and tumor growth and metastatic potential were determined, along with analysis of immune cell populations and expression of epithelial-mesenchymal transition (EMT) markers. Whole-body fractionated-LDI decreased tumor development and lung metastasis not only by infiltration of CD4+, CD8+ T-cells, and dendritic cells (DCs) but also by attenuating EMT. Moreover, a combination of whole-body LDI with localized high-dose radiation therapy reduced the non-irradiated abscopal tumor growth and increased infiltration of effector T cells and DCs. Therefore, whole-body LDI in combination with high-dose radiation therapy could be a potential therapeutic strategy for treating cancer.

Preventative and Therapeutic Effects of Low-dose Ionizing Radiation on the Allergic Response of Rat Basophilic Leukemia Cells.

The prevalence of allergies has increased over the last four decades. In allergic reactions, mast cells induce a hypersensitive immune response to a substance that is normally harmless. Ionizing radiation has different biological effects depending on the dose and dose rate. In this study, we investigated whether low-dose irradiation before (preventative effect) or after (therapeutic effect) an antigen-antibody reaction has an anti-allergic effect. To test this, we activated rat basophilic leukemia (RBL-2H3) mast cells with anti-2,4-dinitrophenyl IgE (antibody) and 2,4-dinitrophenyl human serum albumin, which served as an antigen. To test for both the potential of a preventative effect and a therapeutic effect, we irradiated mast cells both before and after mast cell activation, and we measured mediator release and signaling pathway activity. Low-dose ionizing radiation suppressed mediator release from RBL-2H3 mast cells activated by the antigen-antibody reaction regardless of when the mast cells were irradiated. These results were due to the suppression of FcεRI expression. Therefore, we suggest that low-dose ionizing radiation has a preventative and therapeutic effect in allergic reactions via the FcεRI-mediated RBL-2H3 mast cell activation system.

Low dose radiation attenuates inflammation and promotes wound healing in a mouse burn model.

BACKGROUND: Burn injuries are devastating traumas that functionally affect a variety of organ systems. As intensive inflammatory responses induced by burns can lead to multiple organ failures and impaired skin regeneration increases risk of infectious complex, multimodal therapeutic approaches are needed. OBJECTIVES: To investigate the role of low dose radiation (LDR) treatment for regulation of excessive inflammation and wound healing after burn injury. METHODS: Mouse burn model was established by generating third-degree burn injury in dorsal skin and local LDR less than 100 mGy was delivered to the mice. After 3 or 12 days after burn injury, systemic inflammation in liver, lung, spleen, and kidney and skin wound healing were assessed. For investigation of molecular mechanisms, HaCaT keratinocytes were administrated with serum from mice with burn injury and alteration of viability and cornification biomarkers are assessed. RESULTS: In a mouse burn model, expression of proinflammatory cytokines, interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α, were downregulated by LDR in major organs and wound healing capacity was increased by LDR. In skin tissue, we observed the alleviation of reactive oxygen species generation and increased antioxidant gene expression by LDR. In addition, we found that treatment of serum from mice with burn injury and LDR increased proliferation and cornification in HaCaT cells through activation of focal adhesion kinase signaling pathway. CONCLUSION: LDR could reduce proinflammatory signaling pathway and increase skin wound healing after burn injury. Therefore, the present study suggested LDR as a novel treatment for burn injury patients.

Low-dose ionizing radiation alleviates Aß42-induced cell death via regulating AKT and p38 pathways in Drosophila Alzheimer's disease models.

Ionizing radiation is widely used in medicine and is valuable in both the diagnosis and treatment of many diseases. However, its health effects are ambiguous. Here, we report that low-dose ionizing radiation has beneficial effects in human amyloid-ß42 (Aß42)-expressing Drosophila Alzheimer's disease (AD) models. Ionizing radiation at a dose of 0.05 Gy suppressed AD-like phenotypes, including developmental defects and locomotive dysfunction, but did not alter the decreased survival rates and longevity of Aß42-expressing flies. The same dose of γ-irradiation reduced Aß42-induced cell death in Drosophila AD models through downregulation of head involution defective (hid), which encodes a protein that activates caspases. However, 4 Gy of γ-irradiation increased Aß42-induced cell death without modulating pro-apoptotic genes grim, reaper and hid The AKT signaling pathway, which was suppressed in Drosophila AD models, was activated by either 0.05 or 4 Gy γ-irradiation. Interestingly, p38 mitogen-activated protein-kinase (MAPK) activity was inhibited by exposure to 0.05 Gy γ-irradiation but enhanced by exposure to 4 Gy in Aß42-expressing flies. In addition, overexpression of phosphatase and tensin homolog (PTEN), a negative regulator of the AKT signaling pathway, or a null mutant of AKT strongly suppressed the beneficial effects of low-dose ionizing radiation in Aß42-expressing flies. These results indicate that low-dose ionizing radiation suppresses Aß42-induced cell death through regulation of the AKT and p38 MAPK signaling pathways, suggesting that low-dose ionizing radiation has hormetic effects on the pathogenesis of Aß42-associated AD.

Low-dose ionizing radiation attenuates mast cell migration through suppression of monocyte chemoattractant protein-1 (MCP-1) expression by Nr4a2.

Purpose: The aim of this study was to investigate whether low-dose ionizing radiation attenuates mast cell migration by modulating migration-associated signaling pathways and the expression of chemotactic cytokines.Materials and methods: IgE-sensitized RBL-2H3 mast cells were exposed with ionizing radiation at 0.01, 0.05, 0.1, or 0.5 Gy using a 137Cs γ-irradiator and stimulated with 2,4-dinitrophenol-human serum albumin. Cell migration was determined using a transwell assay system, F-actin distribution using Alex Fluor 488-conjugated phalloidin, expression of various signaling proteins by Western blotting, mRNA expression by RT-PCR.Results: Low-dose ionizing radiation significantly suppressed mast cell migration induced by IgE-mediated mast cell activation. Furthermore, low-dose ionizing radiation altered cell morphology, as reflected by changes in F-actin distribution, and inhibited the activation of PI3K, Btk, Rac1, and Cdc42. These effects were mediated by Nr4a2, an immune-modulating factor. Knockdown of Nr4a2 reduced mast cell migration, inhibited the PI3K and Btk signaling pathways, and reduced expression of the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1). We further demonstrated that direct blockade of MCP-1 using neutralizing antibodies inhibits mast cell migration.Conclusion: Low-dose ionizing radiation inhibits mast cell migration through the regulation production of MCP-1 by Nr4a2 in the activated mast cell system.

A hypoxia-independent up-regulation of hypoxia-inducible factor-1 by AKT contributes to angiogenesis in human gastric cancer.

Underlying mechanisms involved in the activation of hypoxia-inducible factor-1 (HIF-1) in cancer cells are diverse and cell type specific. Although both HIF-1alpha and AKT (protein kinase B) have been implicated in gastric tumor promotion and angiogenesis, it remains unclear whether HIF-1 mediates the role of AKT in terms of promoting vascular endothelial growth factor (VEGF) expression. The present study was performed to investigate the correlation between HIF-1alpha activation and AKT activation in gastric cancer using human gastric cancer specimens, in vitro cell experiments and in vivo animal experiments. Immunohistochemistry performed on tissue array slides containing 268 surgical specimens of gastric carcinomas showed immunoreactivity for HIF-1alpha in 29% of samples. Moreover, HIF-1alpha was positively associated with phosphorylated AKT (pAKT) (P = 0.002) or VEGF (P = 0.002), and the immunoreactivities of pAKT and VEGF were positively correlated (P < 0.001). Western blot analysis and reverse transcription-polymerase chain reaction in cell experiments revealed that the over-expression of constitutively active AKT (CA-AKT) promotes the expressions of HIF-1alpha protein and VEGF messenger ribonucleic acid in Seoul national university (SNU)-216 and SNU-668 gastric cancer cells under normoxic conditions, whereas kinase-dead mutant of AKT down-regulated these expressions under the same conditions. Xenografts in nude mice derived from stable gastric cancer cells over-expressing CA-AKT showed higher tumor incidence, larger tumor volumes, higher microvessel density and stronger HIF-1alpha immunoreactivity than those derived from vector control cells. Thus, we propose that the hypoxia-independent promotion of the AKT-HIF-1alpha-VEGF pathway contributes, at least in part, to gastric cancer tumorigenesis and angiogenesis.

Nuclear factor-kappaB dependency of doxorubicin sensitivity in gastric cancer cells is determined by manganese superoxide dismutase expression.

The role of nuclear factor-kappaB (NF-kappaB) activation in cancer cell apoptosis appears to be tailored specifically for each cell type and the type of NF-kappaB inducer. The present study aimed to determine whether or not NF-kappaB activation is associated with chemosensitivity to doxorubicin (DOX) using the DOX-sensitive SNU-601 and DOX-resistant SNU-216 gastric cancer cell lines. The effect of NF-kappaB activation on DOX (1 microg/mL) sensitivity was analyzed after the suppression of NF-kappaB activation using transfection of the super-suppressive mutant form of IkappaBalpha (mIkappaBalpha) or pretreatment with pyrrolidine dithiocarbamate. In addition, the association between NF-kappaB and manganese superoxide dismutase (MnSOD) in relation to DOX sensitivity was analyzed after the modulation of MnSOD expression. The NF-kappaB activity was much higher in DOX-resistant SNU-216 cells than in DOX-sensitive SNU-601 cells before and after DOX treatment. Overexpression of mIkappaBalpha or pyrrolidine dithiocarbamate pretreatment decreased the DOX resistance in SNU-601 cells with low MnSOD expression, but not in SNU-216 cells with high MnSOD expression. In comparison, the overexpression of MnSOD, which also suppressed NF-kappaB activation in both cell lines, increased DOX resistance in SNU-601 cells. Blocking of MnSOD expression using RNA interference techniques increased DOX sensitivity in SNU-216 cells, which was further augmented by the additional inhibition of NF-kappaB activity. Our results showed that whether NF-kappaB contributes to DOX sensitivity in gastric cancer cells is determined by the level of MnSOD expression. Thus, targeting both MnSOD and NF-kappaB may be helpful for increasing the efficacy of DOX treatment of DOX-resistant SNU gastric cancer cells.

Comprehensive analysis of time- and dose-dependent patterns of gene expression in a human mesenchymal stem cell line exposed to low-dose ionizing radiation.

We focused on the transcriptional responses induced by low and very low doses of ionizing radiation with time effect. Regardless of their importance only a few limited studies have been done. Here we applied a large-scale gene transcript profile to elucidate the genes and biological pathways. Immortalized human mesenchymal stem cells were irradiated with 0.01, 0.05, 0.2 and 1 Gy of gamma radiation and total RNA was extracted from each cell line at 1, 4, 12 and 48 h after exposure. The essential transcriptional responses were identified according to dose and time. A total of 6,016 genes showed altered expression patterns at more than one time point or dose level among the investigated 10,800 genes. Genes that showed dose-dependent expression responses were involved in signal transduction, regulation of transcription, proteolysis, peptidolysis and metabolism. Those that showed time-dependent responses were divided into two distinct groups: the up-and-down group was associated with 'cellular defense mechanisms' such as apoptosis, cell adhesion, stress response and immune response and the down-and-up group with 'fundamental cellular processes' such as DNA replication, mitosis, RNA splicing, DNA repair and translation initiation. Genes showing both dose-and time-dependent responses exhibited a mixture of both features. A highly non-linear relationship between the IR dose and the transcriptional relative response was obtained from the dose-dependent group. The time-dependent group also exhibited a non-linear relationship as the complex effect group did. Some of the early-reactive-phase (1-4 h) genes showed a differential expression response to 0.01, 0.05 and 0.2 Gy but were unresponsive to 1 Gy. Some of the late-recovery-phase (12-48 h) genes showed a differential expression to 1 Gy but were relatively unresponsive to other doses. We further characterized the gene expression patterns that could be implicated in the molecular mechanism of the cellular responses to low and very low-dose irradiation.