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Summary The selective depletion of senescent cells (SCs) by small molecules, termed senolytic agents, is a promising therapeutic approach for treating age‐related diseases and chemotherapy‐ and radiotherapy‐induced side effects. Piperlongumine (PL) was recently identified as a novel senolytic agent. However, its mechanism of action and molecular targets in SCs was unknown and thus was investigated. Specifically, we used a PL‐based chemical probe to pull‐down PL‐binding proteins from live cells and then mass spectrometry‐based proteomic analysis to identify potential molecular targets of PL in SCs. One prominent target was oxidation resistance 1 (OXR1), an important antioxidant protein that regulates the expression of a variety of antioxidant enzymes. We found that OXR1 was upregulated in senescent human WI38 fibroblasts. PL bound to OXR1 directly and induced its degradation through the ubiquitin‐proteasome system in an SC‐specific manner. The knockdown of OXR1 expression by RNA interference significantly increased the production of reactive oxygen species in SCs in conjunction with the downregulation of antioxidant enzymes such as heme oxygenase 1, glutathione peroxidase 2, and catalase, but these effects were much less significant when OXR1 was knocked down in non‐SCs. More importantly, knocking down OXR1 selectively induced apoptosis in SCs and sensitized the cells to oxidative stress caused by hydrogen peroxide. These findings provide new insights into the mechanism by which SCs are highly resistant to oxidative stress and suggest that OXR1 is a novel senolytic target that can be further exploited for the development of new senolytic agents.

There is an increasing need to better understand the long-term health effects of high-linear energy transfer (LET) radiation due to exposure during space missions, as well as its increasing use in clinical treatments. Previous studies have indicated that exposure to 56Fe heavy ions increases the incidence of acute myeloid leukemia (AML) in mice but the underlying molecular mechanisms remain elusive. Epigenetic alterations play a role in radiation-induced genomic instability and the initiation and progression of AML. In this study, we assessed the effects of low-dose 56Fe-ion irradiation on epigenetic alterations in bone marrow mononuclear cells (BM-MNCs) and hematopoietic progenitor and stem cells (HPSCs). Exposure to 56Fe ions (600 MeV, 0.1, 0.2 and 0.4 Gy) resulted in significant epigenetic alterations involving methylation of DNA, the DNA methylation machinery and expression of repetitive elements. Four weeks after irradiation, these changes were primarily confined to HPSCs and were exhibited as dose-dependent hypermethylation of LINE1 and SINE B1 repetitive elements [4.2-fold increase in LINE1 (P < 0.001) and 7.6-fold increase in SINE B1 (P < 0.01) after exposure to 0.4 Gy; n = 5]. Epigenetic alterations were persistent and detectable for at least 22 weeks after exposure, when significant loss of global DNA hypomethylation (1.9-fold, P < 0.05), decreased expression of Dnmt1 (1.9-fold, P < 0.01), and increased expression of LINE1 and SINE B1 repetitive elements (2.8-fold, P < 0.001 for LINE1 and 1.9-fold, P < 0.05 for SINE B1; n = 5) were observed after exposure to 0.4 Gy. In contrast, exposure to 56Fe ions did not result in accumulation of increased production of reactive oxygen species (ROS) and DNA damage, exhibited as DNA strand breaks. Furthermore, no significant alterations in cellular senescence and apoptosis were detected in HPSCs after exposure to 56Fe-ion radiation. These findings suggest that epigenetic reprogramming is possibly involved in the development of radiation-induced genomic instability and thus, may have a causative role in the development of AML.

Due to improvements in early detection and treatment of cancer, the number of long-term cancer survivors is increasing. Unfortunately, these survivors are at increased risk for developing cancer treatment-related late effects, including ionizing radiation (IR)- and chemotherapy-induced long-term bone marrow (LT-BM) injury. Because LT-BM injury can deteriorate over time or after the patients receiving additional cancer treatment or undergoing autologous BM transplantation, it may eventually lead to the development of hypoplastic anemia or myelodysplastic syndrome. This review is to provide a survey of some of these recent findings regarding the underlying mechanisms by which IR and chemotherapy cause LT-BM injury. Particularly, we will highlight the discoveries of the role of reactive oxygen species in regulating HSC self-renewal and the role of oxidative stress in mediating IR- and chemotherapy-induced HSC senescence and LT-BM injury. These discoveries may lead to the development of new therapeutic strategies that have the potential to reduce the late adverse effects of conventional cancer therapy on the hematopoietic system in long-term cancer survivors.

Our study was to elucidate the mechanisms whereby BMS-345541 (BMS, a specific IκB kinase β inhibitor) inhibits the repair of DNA double-strand breaks (DSBs) and evaluate whether BMS can sensitize MCF-7 breast cancer cells (MCF-7 cells) to ionizing radiation (IR) in an apoptosis-independent manner. In this study, MCF-7 cells were exposed to IR in vitro and in vivo with or without pretreatment of BMS. The effects of BMS on the repair of IR-induced DSBs by homologous recombination (HR) and non-homologous end-joining (NHEJ) were analyzed by the DR-GFP and EJ5-GFP reporter assays and IR-induced γ-H2AX, 53BP1, Brca1 and Rad51 foci assays. The mechanisms by which BMS inhibits HR were examined by microarray analysis and quantitative reverse transcription PCR. The effects of BMS on the sensitivity of MCF-7 cells to IR were determined by MTT and clonogenic assays in vitro and tumor growth inhibition in vivo in a xenograft mouse model. The results showed that BMS selectively inhibited HR repair of DSBs in MCF-7 cells, most likely by down-regulation of several genes that participate in HR. This resulted in a significant increase in the DNA damage response that sensitizes MCF-7 cells to IR-induced cell death in an apoptosis-independent manner. Furthermore, BMS treatment sensitized MCF-7 xenograft tumors to radiation therapy in vivo in an association with a significant delay in the repair of IR-induced DSBs. These data suggest that BMS is a novel HR inhibitor that has the potential to be used as a radiosensitizer to increase the responsiveness of cancer to radiotherapy.

miRNAs are small, non-coding RNAs that negatively regulate gene expression at the post-transcriptional and translational levels. miRNA-451 was previously reported to be down-regulated in gastric and colorectal cancers. Here, we showed that miRNA-451 expression decreased in non-small cell lung cancer (NSCLC) tissues and that its expression was negatively associated with lymph node metastasis, the stage of TNM classification and poor prognosis of NSCLC patients. Moreover, significantly different miRNA-451 expression levels were found between smoking and non-smoking patients. The overexpression of miRNA-451 inhibited cell cycle progression, cellular migration and the invasive ability of NSCLC cells. Increased miRNA-451 expression also promoted anoikis of NSCLC cells. Together, these data suggested that aberrantly expressed miRNA-451 may be associated with the development of NSCLC.

p38 mitogen-activated protein kinases (p38) has been shown to be activated in hematopoietic stem and progenitors cells after exposure to ionizing radiation (IR) and its activation has been implicated in bone marrow (BM) suppression under various pathological conditions. Therefore, in the present study we investigated whether inhibition of p38 activity alone with SB203580 (SB, a specific p38 inhibitor) or in combination with granulocyte colony-stimulating factor (G-CSF) can mitigate total body irradiation (TBI)-induced BM damage and lethality. Our results showed that p38 inhibition with SB had no significant effect on the 30-day survival rates of the mice exposed to 7.2 Gy TBI when it was used alone but increased the survival of the mice when it was combined with G-CSF. This combined effect may be attributable to a better preservation or stimulation of hematopoietic stem and progenitor cells, because BM cells from SB and G-CSF-treated mice produced more colony forming units-granulocyte-macrophage (CFU-GM) and 4-week cobblestone area forming cells (CAFCs) than the cells from either SB or G-CSF-treated mice after TBI in a colony forming cell assay and a CAFC assay, respectively. These findings suggest that the combined therapy with SB and G-GSF is more effective in mitigating TBI-induced acute BM injury than either agent alone.

Xiao-Dong Zhang*, Hong-Ying Wu*, Di Wu, Yue-Ying Wang, Jian-Hui Chang, Zhi-Bin Zhai, Ai-Min Meng, Pei-Xun Liu, Liang-An Zhang, Fei-Yue FanInstitute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, and Tianjin Key Laboratory of Molecular Nuclear Medicine, Tianjin, People’s Republic of China *The authors contributed equally to this workAbstract: Gold nanoparticles have potential applications in biomedicine, but one of the important concerns is about their safety. Most toxicology data are derived from in vitro studies and may not reflect in vivo responses. Here, an animal toxicity study of 13.5 nm gold nanoparticles in mice is presented. Animal survival, weight, hematology, morphology, and organ index are characterized at different concentrations (137.5–2200 µg/kg) over 14–28 days. The results show that low concentrations of gold nanoparticles do not cause an obvious decrease in body weight or appreciable toxicity, even after their breakdown in vivo. High concentrations of gold nanoparticles induced decreases in body weight, red blood cells, and hematocrit. It was also found that gold nanoparticles administered orally caused significant decreases in body weight, spleen index, and red blood cells. Of the three administration routes, the oral and intraperitoneal routes showed the highest toxicity, and the tail vein injection showed the lowest toxicity. Combining the results of all of these studies, we suggest that targeted gold nanopartices by tail vein injection may be suitable for enhancement of radiotherapy, photothermal therapy, and related medical diagnostic procedures.Keywords: gold nanoparticles, in vivo, toxicity

Macrophages (MΦ) are professional phagocytes in the innate immune system. They are not only involved in regulation of various immune functions and inflammation, but also exhibit plasticity in modulation of tissue regeneration and repair after being polarized into M1 and M2 MΦ by different inflammatory cytokines. In addition, several recent studies show that MΦ are a new constituent of the hematopoietic stem cells (HSCs) niche and play a role in regulation of HSCs maintenance and mobilization in bone marrow (BM). However, it is not known whether MΦ can regulate HSCs self-renewal and whether the effects of MΦ on HSCs can be influenced by differential MΦ polarization. This was investigated using an ex vivo HSCs expansion model consisting of mouse bone marrow LSK (Lin-sca-1+c-Kit+) cells cultured with or without MΦ in a mouse HSCs expansion medium (StemSpanTM serum-free medium supplemented with 20ng/ml of stem cell factor [SCF] and thrombopoietin [TPO]). We found that LSK cells were expanded about 20-, 15-, and 30-fold after 6 days of co-culture with MΦ harvested from mouse BM, spleen, and peritoneal cavity, respectively, whereas there was no significant expansion after culture without MΦ or with BM Gr-1high or Gr-1low monocytes. In addition, we found that M1-MΦ polarized by INFγ were more effective than IL4-polarized M2-MΦ in promoting LSK cells expansion ex vivo (45-fold vs. 15-fold). However, the promotion of LSK cells expansion by M1-MΦ resulted in about 88% reduction in HSCs as judged by 5-week cobblestone area forming cell (CAFC) assay. In contrast, M2-MΦ significantly promoted HSCs expansion. A greater expansion of HSCs was achieved after LSK cells were co-cultured with M2-MΦ for 9 days than for 6 days (20-fold vs. 6-fold). These findings suggest that M1-MΦ are more effective than M2-MΦ in promoting LSK cells or hematopoietic progenitor cells (HPCs) expansion, at the expense of HSCs self-renewal, whereas M2-MΦ can promote HPCs expansion as well as HSCs self-renewal. This suggestion is supported by results of serial transplantation and competitive repopulation unit (CRU) assays. CRU assay showed that LT-HSCs (e.g. 4-month CRU) were increased about 13 folds relative to the starting numbers of CRU in the input after LSK cells were co-cultured with M2-MΦ for 9 days, but were barely detectable after the cells were cultured without MΦ or with M1-MΦ. The inhibitory effect of M1-MΦ on HSCs self-renewal and expansion was attenuated by inhibition of inducible nitric oxide synthase (iNOS) activity with an inhibitor or knockout iNOS. Inhibition of arginase and/or cyclooxygenase activities with an inhibitor attenuated the promotion of HSCs self-renewal and expansion by M2-MΦ. More importantly, we found that human CD34+ cells, 8-week CAFC, and SCID mice repopulating cells (SRCs) were increased 42±14, 8±2.1, and 4 folds over the input values, respectively, after human cord blood CD34+ cells were co-cultured with M2-MΦ generated from human cord blood CD34- cells for 7 days in a human HSCs expansion medium (StemSpanTM serum-free medium supplemented with 50 ng/ml of SCF, TPO, and FLT-3 ligand). These findings demonstrate that M1-MΦ and M2-MΦ have opposite effects on HSCs self-renewal, which may be important for regulation of hematopoiesis under various pathological conditions in which MΦ are differentially polarized to M1 or M2 by diverse inflammatory cytokines. In addition, M2-MΦ may be used to promote human cord blood HSCs ex vivo expansion to make human cord blood transplantation available to more patients. Disclosures No relevant conflicts of interest to declare.

Knowledge of the mechanisms involved in the radiation response is critical for developing interventions to mitigate radiation-induced injury to normal tissues. Exposure to radiation leads to increased oxidative stress, DNA-damage, genomic instability and inflammation. The transcription factor CCAAT/enhancer binding protein delta (Cebpd; C/EBPδ is implicated in regulation of these same processes, but its role in radiation response is not known. We investigated the role of C/EBPδ in radiation-induced hematopoietic and intestinal injury using a Cebpd knockout mouse model. Cebpd−/− mice showed increased lethality at 7.4 and 8.5 Gy total-body irradiation (TBI), compared to Cebpd+/+ mice. Two weeks after a 6 Gy dose of TBI, Cebpd−/− mice showed decreased recovery of white blood cells, neutrophils, platelets, myeloid cells and bone marrow mononuclear cells, decreased colony-forming ability of bone marrow progenitor cells, and increased apoptosis of hematopoietic progenitor and stem cells compared to Cebpd+/+ controls. Cebpd−/− mice exhibited a significant dose-dependent decrease in intestinal crypt survival and in plasma citrulline levels compared to Cebpd+/+ mice after exposure to radiation. This was accompanied by significantly decreased expression of γ-H2AX in Cebpd−/− intestinal crypts and villi at 1 h post-TBI, increased mitotic index at 24 h post-TBI, and increase in apoptosis in intestinal crypts and stromal cells of Cebpd−/− compared to Cebpd+/+ mice at 4 h post-irradiation. This study uncovers a novel biological function for C/EBPδ in promoting the response to radiation-induced DNA-damage and in protecting hematopoietic and intestinal tissues from radiation-induced injury.