Sequential Treatment by Ionizing Radiation and Sodium Arsenite Dramatically Accelerates TRAIL-Mediated Apoptosis of Human Melanoma Cells

Melanoma is the most lethal form of skin cancer. There is a lack of effective treatments for individuals with advanced disease. Many melanomas exhibit high levels of radioresistance. The direct consequence of γ-irradiation for most melanoma cells is growth arrest at the G2-M phase of cell cycle. However, radiation-induced signaling pathways may affect numerous additional targets in cancer cells. We show in the present study that γ-irradiation, as well as α-particle exposure, dramatically increases the susceptibility of melanoma cells to recombinant tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-mediated apoptosis via up-regulation of surface TRAIL-receptor 1/receptor 2 (DR4/DR5) levels and to Fas ligand–mediated apoptosis via up-regulation of surface Fas levels. Additionally, increased dynamin-2 expression after irradiation is critically important in the translocation of death receptor to the cell surface. Moreover, sodium arsenite treatment may up-regulate expression of endogenous TRAIL and induces its translocation to cell surface and further down-regulates cFLIP levels in melanoma cells. We have evaluated the effects of sequential γ-irradiation and arsenite treatment of melanoma cells for the induction of death signaling. Such treatment results in an efficient TRAIL-mediated apoptosis via a paracrine mechanism. These data highlight the efficacy of combined modality treatment involving radiation and arsenite in clinical management of this often fatal form of skin cancer

Molecular detection of Torque teno virus in different breeds of swine

<p>Abstract</p> <p>Background</p> <p>Torque teno virus (TTV), of the <it>Anelloviridae </it>family, <it>Iotatorquevirus </it>genus, is a non-enveloped, single-stranded, and negative sense DNA (ssDNA) virus infecting human and many domestic animals including swines. Very little information is known about the investigations of TTV prevalence in different swine breeds so far.</p> <p>Methods</p> <p>In this study, 208 serum samples collected from seven swine breeds (<it>Rongchang pig</it>, <it>Chenghua pig</it>, <it>Zibet pig</it>, <it>Wild boar</it>, <it>Duroc</it>, <it>Landrace</it>, <it>Large Yorkshire</it>) from two independent farms were detected to determine the prevalence of two swine TTV genogroups, TTV1 and TTV 2, by nested polymerase chain reaction methods, and to analyse prevalence difference among these breeds.</p> <p>Results</p> <p>The results showed that the prevalence of TTV in the seven breeds was 92%-100%. No significant difference (p > 0.05) in TTV infection was observed between different breeds. Interestingly, significantly higher prevalence for TTV1 in <it>Rongchang </it>boars (90%) and for TTV2 in <it>Rongchang </it>sows (95%) were detected, while co-infection rate (43.8%) was lower than other breeds. Sequence analysis showed that the homology of TTV1 and TTV2 were over 90.9% and 86.4% in these breeds, respectively.</p> <p>Conclusions</p> <p>The results indicated that TTV was widely distributed in the seven swine breeds. The prevalence of both TTV genogroups associated with swine breeds and genders. This study also respented the first description of swine TTV prevalence in different swine breeds. It was vitally necessary to further study swine TTV pathogenicity.</p

Inhibition of Ataxia Telangiectasia Mutated Kinase Activity Enhances TRAIL-Mediated Apoptosis in Human Melanoma Cells

The aim of the present study was to elucidate the effects of ataxia telangiectasia mutated (ATM) kinase on the regulation of the extrinsic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2/DR5-mediated death pathway in human melanoma cells. We revealed that total ATM protein levels were high in some human melanoma lines compared with normal cells. The basal levels of active form ATM phospho-Ser1981 were also detectable in many melanoma lines and could be further up-regulated by γ-irradiation. Pretreatment of several melanoma lines just before γ-irradiation with the inhibitor of ATM kinase KU-55933 suppressed p53 and nuclear factor-κB (NF-κB) activation but notably increased radiation-induced DR5 surface expression, down-regulated cFLIP (caspase-8 inhibitor) levels, and substantially enhanced exogenous TRAIL-induced apoptosis. Furthermore, γ-irradiation in the presence of KU-55933 rendered TRAIL-resistant HHMSX melanoma cells susceptible to TRAIL-mediated apoptosis. In addition, suppression of ATM expression by the specific short hairpin RNA also resulted in down-regulation of cFLIP levels, up-regulation of surface DR5 expression, and TRAIL-mediated apoptosis in melanoma cells. Besides p53 and NF-κB, crucial regulators of DR5 expression, transcription factor STAT3 is known to negatively regulate DR5 expression. Suppression of Ser727 and Tyr705 phosphorylation of STAT3 by KU-55933 reduced STAT3 transacting activity accompanied by elevation in DR5 expression. Dominant-negative STAT3β also efficiently up-regulated the DR5 surface expression and down-regulated cFLIP levels in melanoma cells in culture and in vivo. Taken together, our data show the existence of an ATM-dependent STAT3-mediated antiapoptotic pathway, which on suppression sensitizes human melanoma cells to TRAIL-mediated apoptosis

Radiation Induced Non-Targeted Response: Mechanism and Potential Clinical Implications

Generations of students in radiation biology have been taught that heritable biological effects require direct damage to DNA. Radiation-induced non-targeted/bystander effects represent a paradigm shift in our understanding of the radiobiological effects of ionizing radiation in that extranuclear and extracellular effects may also contribute to the biological consequences of exposure to low doses of radiation. Although radiation induced bystander effects have been well documented in a variety of biological systems, including 3D human tissue samples and whole organisms, the mechanism is not known. There is recent evidence that the NF-κB-dependent gene expression of interleukin 8, interleukin 6, cyclooxygenase- 2, tumor necrosis factor and interleukin 33 in directly irradiated cells produced the cytokines and prostaglandin E2 with autocrine/paracrine functions, which further activated signaling pathways and induced NF-κB-dependent gene expression in bystander cells. The observations that heritable DNA alterations can be propagated to cells many generations after radiation exposure and that bystander cells exhibit genomic instability in ways similar to directly hit cells indicate that the low dose radiation response is a complex interplay of various modulating factors. The potential implication of the non-targeted response in radiation induced secondary cancer is discussed. A better understanding of the mechanism of the non-targeted effects will be invaluable to assess its clinical relevance and ways in which the bystander phenomenon can be manipulated to increase therapeutic gain in radiotherapy

Mitochondrial Function and Nuclear Factor-KB–Mediated Signaling in Radiation-Induced Bystander Effects

Although radiation-induced bystander effects have been well described over the past decade, the mechanisms of the signaling processes involved in the bystander phenomenon remain unclear. In the present study, using the Columbia University charged particle microbeam, we found that mitochondrial DNA–depleted human skin fibroblasts (ρo) showed a higher bystander mutagenic response in confluent monolayers when a fraction of the same population were irradiated with lethal doses compared with their parental mitochondrial–functional cells (ρ+). However, using mixed cultures of ρo and ρ+ cells and targeting only one population of cells with a lethal dose of α-particles, a decreased bystander mutagenesis was uniformly found in nonirradiated bystander cells of both cell types, indicating that signals from one cell type can modulate expression of bystander response in another cell type. In addition, we found that Bay 11-7082, a pharmacologic inhibitor of nuclear factor-κB (NF-κB) activation, and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a scavenger of nitric oxide (NO), significantly decreased the mutation frequency in both bystander ρo and ρ+ cells. Furthermore, we found that NF-κB activity and its dependent proteins, cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS), were lower in bystander ρo cells when compared with their ρ+ counterparts. Our results indicated that mitochondria play an important role in the regulation of radiation-induced bystander effects and that mitochondria-dependent NF-κB/iNOS/NO and NF-κB/COX-2/prostaglandin E2 signaling pathways are important to the process

Radiation Response and Regulation of Apoptosis Induced by a Combination of TRAIL and CHX in Cells Lacking Mitochondrial DNA: A Role for NF-KB–STAT3-directed Gene Expression

Mitochondrial DNA depleted (ρ0) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-κB and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental ρ+ HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in ρ0 cells compared to ρ+ HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, ΙL17Β, ΙL18, ΙL19, and ΙL28Β) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-κB and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-κB/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in ρ+ HSF, but this response was substantially decreased in ρ0 HSF. Suppression of the IKK–NF-κB pathway by the small molecular inhibitor BMS-345541 and of the JAK2–STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated ρ+ HSF. Inhibitory antibodies against IL6, the main activator of JAK2–STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-κB activation was partially lost in ρ0 HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-κB targets, further suppressing IL6–JAK2–STAT3 that in concert with NF-κB regulated protection against TRAIL-induced apoptosis

Resveratrol Sensitizes Melanomas to TRAIL Through Modulation of Antiapoptotic Gene Expression

Although many human melanomas express the death receptors TRAIL-R2/DR5 or TRAIL-R1/DR4 on cell surface, they often exhibit resistance to exogenous TRAIL. One of the main contributors to TRAIL-resistance of melanoma cells is upregulation of transcription factors STAT3 and NF-κB that control the expression of antiapoptotic genes, including cFLIP and Bcl-xL. On the other hand, the JNK-cJun pathway is involved in the negative regulation of cFLIP (a caspase-8 inhibitor) expression. Our observations indicated that resveratrol, a polyphenolic phytoalexin, decreased STAT3 and NF-κB activation, while activating JNK-cJun that finally suppressed expression of cFLIP and Bcl-xL proteins and increased sensitivity to exogenous TRAIL in DR5-positive melanomas. Interestingly, resveratrol did not increase surface expression of DR5 in human melanomas, while γ-irradiation or sodium arsenite treatment substantially upregulated DR5 expression. Hence, an initial increase in DR5 surface expression (either by γ-irradiation or arsenite), and subsequent downregulation of antiapoptotic cFLIP and Bcl-xL (by resveratrol), appear to constitute an efficient approach to reactivate apoptotic death pathways in TRAIL-resistant human melanomas. In spite of partial suppression of mitochondrial function and the mitochondrial death pathway, melanoma cells still retain the potential to undergo the DR5-mediated, caspase-8-dependent death pathway that could be accelerated by either an increase in DR5 surface expression or suppression of cFLIP. Taken together, these results suggest that resveratrol, in combination with TRAIL, may have a significant efficacy in the treatment of human melanomas

Radiation-Induced Bystander Signaling Pathways in Human Fibroblasts: A Role for Interleukin-33 in the Signal Transmission

The main goal of this study is to elucidate the mechanisms of the signal transmission for radiation-induced bystander response. The NF-κB-dependent gene expression of IL8, IL6, PTGS2/COX2, TNF and IL33 in directly irradiated human skin fibroblasts produced the cytokines and prostaglandin E2 (PGE2) with autocrine/paracrine functions, which further activated signaling pathways and induced NF-κB-dependent gene expression in bystander cells. As a result, bystander cells also started expression and production of interleukin-8, interleukin-6, COX-2-generated PGE2 and interleukin-33 (IL-33) followed by autocrine/paracrine stimulation of the NF-κB and MAPK pathways. A blockage of IL-33 transmitting functions with anti-IL-33 monoclonal antibody added into the culture media decreased NF-κB activation in directly irradiated and bystander cells. On the other hand, the IGF-1-Receptor kinase regulated the PI3K–AKT pathway in both directly irradiated and bystander fibroblasts. A pronounced and prolonged increase in AKT activity after irradiation was a characteristic feature of bystander cells. AKT positively regulated IL-33 protein expression levels. Suppression of the IGF-R1–AKT–IL-33 pathway substantially increased radiation-induced or TRAIL-induced apoptosis in fibroblasts. Taken together, our results demonstrated the early activation of NF-κB-dependent gene expression first in directly irradiated and then bystander fibroblasts, the further modulation of critical proteins, including IL-33, by AKT in bystander cells and late drastic changes in cell survival and in enhanced sensitivity to TRAIL-induced apoptosis after suppression of the IGF-1R–AKT–IL-33 signaling cascade in both directly irradiated and bystander cells

Mechanism of Radiation-Induced Bystander Effects: A Unifying Model

The radiation-induced bystander effect represents a paradigm shift in our understanding of the radiobiological effects of ionizing radiation, in that extranuclear and extracellular events may also contribute to the final biological consequences of exposure to low doses of radiation. Although radiation-induced bystander effects have been well documented in a variety of biological systems, the mechanism is not known. It is likely that multiple pathways are involved in the bystander phenomenon, and different cell types respond differently to bystander signalling. Using cDNA microarrays, a number of cellular signalling genes, including cyclooxygenase-2 (COX-2), have been shown to be causally linked to the bystander phenomenon. The observation that inhibition of the phosphorylation of extracellular signal-related kinase (ERK) suppressed the bystander response further confirmed the important role of the mitogen-activated protein kinase (MAPK) signalling cascade in the bystander process. Furthermore, cells deficient in mitochondrial DNA showed a significantly reduced response to bystander signalling, suggesting a functional role of mitochondria in the signalling process. Inhibitors of nitric oxide (NO) synthase (NOS) and mitochondrial calcium uptake provided evidence that NO and calcium signalling are part of the signalling cascade. The bystander observations imply that the relevant target for various radiobiological endpoints is larger than an individual cell. A better understanding of the cellular and molecular mechanisms of the bystander phenomenon, together with evidence of their occurrence in-vivo, will allow us to formulate a more accurate model for assessing the health effects of low doses of ionizing radiation