Protect Bone Marrow Research Articles

Lung and Heart Injury in a Nonhuman Primate Model of Partial-body Irradiation with Minimal Bone Marrow Sparing: Histopathological Evidence of Lung and Heart Injury.

Male rhesus macaques were subjected to partial-body irradiation at 10, 11, or 12 Gy with 5% bone marrow protection. Animals were euthanized when dictated by prospectively determined clinical parameters or at approximately 180 d following irradiation. Histological sections of lung and heart were stained with hematoxylin and eosin as well as a battery of histochemical and immunohistochemical stains. Histopathological alterations in the lung were centered on fibrosis, inflammation, and reactive/proliferative changes in pneumocytes. These changes were noted in animals necropsied after approximately 85-100 d postirradiation and extending through the observation period. Interstitial and pleural fibrosis demonstrated by Masson's trichrome staining were associated with increased alpha smooth muscle actin and collagen 1 immunohistochemical staining. Areas of interstitial fibrosis had reduced microvascular density with CD31 immunohistochemical staining. Accumulations of CD163- and CD206-positive alveolar macrophages were present in areas of interstitial fibrosis. Unidentified cells termed "myxoid" cells in alveolar walls had histochemical and immunohistochemical staining characteristics of epithelial-, endothelial-, or pericyte-mesenchymal transition states that were developing myofibroblast features. Distinctive focal or multifocal alveolar-bronchiolar hyperplasia had microscopic features of preneoplastic proliferation. Delayed radiation-associated changes in the heart consisted primarily of myocardial fibrosis, with rare histological evidence of myofiber degeneration.

Histopathological Features of the Development of Intestine and Mesenteric Lymph Node Injury in a Nonhuman Primate Model of Partial-body Irradiation with Minimal Bone Marrow Sparing.

Male rhesus macaques were subjected to partial-body irradiation at 10, 11, or 12 Gy with 5% bone marrow protection. Animals were euthanized when dictated by prospectively determined clinical parameters or at approximately 180 d following irradiation. Histological sections of jejunum, colon, and mesenteric lymph node were stained with hematoxylin and eosin as well as a battery of histochemical and immunohistochemical stains. The immediate postirradiation histopathological alterations in the jejunum and colon were based primarily on injury to rapidly proliferating crypt epithelial cells, though there was evidence of additional radiation-induced fibrogenic responses. There was substantial resolution of the radiation-related mucosal injury through the observation period, but microscopically visible defects in mucosal structure persisted to the end of the observation period. In the later stages of the observation period, the jejunum and colon had overt fibrosis that was most commonly located in the submucosa and serosa, with less microscopically discernible involvement of the mucosa. Mesenteric lymph nodes had an immediate postirradiation reduction in cellularity due to the known effects of irradiation on lymphoid cell populations. In later stages of the observation period the lymph nodes also developed fibrotic changes, possibly related to transmigration of immunomodulatory cells and/or signaling molecules from the radiation-damaged intestine.

Flavonoid genistein protects bone marrow sinusoidal blood vessels from damage by methotrexate therapy in rats.

Cancer chemotherapy can cause significant damage to the bone marrow (BM) microvascular (sinusoidal) system. Investigations must now address whether and how BM sinusoidal endothelial cells (SECs) can be protected during chemotherapy. Herein we examined the potential protective effects of genistein, a soy-derived flavonoid, against BM sinusoidal damage caused by treatment with methotrexate (MTX). The groups of young adult rats were gavaged daily with genistein (20 mg/kg) or placebo. After 1 week, rats also received daily injections of MTX (0.75 mg/kg) or saline for 5 days and were killed after a further 4 days. Histological analyses showed that BM sinusoids were markedly dilated ( p < 0.001) in the MTX-alone group but were unaffected or less dilated in the genistein+MTX group. In control rats, genistein significantly enhanced expression of vascular endothelial growth factor (VEGF; p < 0.01), particularly in osteoblasts, and angiogenesis marker CD31 ( p < 0.001) in bone. In MTX-treated rats, genistein suppressed MTX-induced apoptosis of BM SECs ( p < 0.001 vs MTX alone group) and tended to increase expression of CD31 and VEGF ( p < 0.05). Our in vitro studies showed that genistein in certain concentrations protected cultured SECs from MTX cytotoxic effects. Genistein enhanced tube formation of cultured SECs, which is associated with its ability to induce expression of endothelial nitric oxide synthase and production of nitric oxide. These data suggest that genistein can protect BM sinusoids during MTX therapy, which is associated, at least partially, with its indirect effect of promoting VEGF expression in osteoblasts and its direct effect of enhancing nitric oxide production in SECs.

Protection Against Radiation-Induced Micronuclei in Rat Bone Marrow Erythrocytes by Curcumin and Selenium L-Methionine.

The search for potent radioprotective agents for the amelioration of radiation side effect is an important aim in radiobiology. The present study aimed to evaluate the effects of curcumin and seleno-L-methionine against radiation-induced micronucleus formation in rat bone marrow. In total, 40 male rats were divided into 8 groups (n=5 each), including control, curcumin or seleno-L-methionine treated alone or in combination, 2 Gy irradiation, irradiation of treated groups with curcumin or seleno-L-methionine or their combination. Curcumin was administrated orally and seleno-L-methionine was injected intraperitoneally 24 hours before irradiation. The frequency of micronucleated normochromatic erythrocytes (MnNCEs) and micronucleated polychromatic erythrocytes (MnPCEs) was scored in 5,000 polychromatic erythrocytes (PCEs) and the cell proliferation ratio [(PCE/(PCE+NCE); NCE=normochromatic erythrocytes] was calculated for each treatment group. Data were analyzed by the SPSS software version 16.0 and P<0.05 was considered as statistically significant differences. Pretreatment with curcumin and seleno-L-methionine before irradiation reduced the frequency of MnPCEs and MnNCEs (P=0.01) and increased the cell proliferation ratio. Moreover, the results showed that this pretreatment reduced the frequency of MnPCEs with a protection factor (PF) of 1.2 and 1.6, respectively. The combination of curcumin and seleno-L-methionine in reducing MnPCEs and MnNCEs was not more effective than each agent alone, while improved cell proliferation ratio. Both curcumin and seleno-L-methionine showed potent protection against radiation induced MN in bone marrow cells. The combination of the two agents further ameliorates this activity, thus leading to improve bone marrow protection.

Radioprotective effects of roxadustat (FG-4592) in haematopoietic system.

BackgroundIonizing radiation often causes severe injuries to radiosensitive tissues, especially haematopoietic system. Novel radioprotective drugs with low toxicity and high effectiveness are required. Prolyl hydroxylases domain (PHD) inhibitors have been reported to protect against radiation‐induced gastrointestinal toxicity. In this study, we demonstrated the protective effects of a PHD inhibitor, roxadustat (FG‐4592), against radiation‐induced haematopoietic injuries in vitro and in vivo.MethodsTissue injuries were evaluated by Haematoxilin‐Eosin (HE) staining assay. HSCs were determined by flow cytometry with the Lin−Sca‐1+c‐Kit+ (LSK) phenotype. Cell apoptosis was determined by Annexin V/PI staining assay. Immunofluorescence was performed to measure radiation‐induced DNA damage. A western blot assay was used to detect the changes of proteins related to apoptosis.ResultsWe found that FG‐4592 pretreatment increased survival rate of irradiated mice and protected bone marrow and spleen from damages. Number of bone marrow cells (BMCs) and LSK cells were also increased both in irradiated mice and recipients after bone marrow transplantation (BMT). FG‐4592 also protected cells against radiation‐induced apoptosis and double strand break of DNA.ConclusionsOur data showed that FG‐4592 exhibited radioprotective properties in haematopoietic system both in vivo and in vitro through up‐regulating HIF‐1α, indicating a potential role of FG‐4592 as a novel radioprotector.

The specific role of O6-methylguanine-DNA methyltransferase inhibitors in cancer chemotherapy.

The DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT), can confer resistance to guanine O6-alkylating agents. Therefore, inhibition of resistant MGMT protein is a practical approach to increase the anticancer effects of such alkylating agents. Numerous small molecule inhibitors were synthesized and exhibited potential MGMT inhibitory activities. Although they were nontoxic alone, they also inhibited MGMT in normal tissues, thereby enhancing the side effects of chemotherapy. Therefore, strategies for tumor-specific MGMT inhibition have been proposed, including local drug delivery and tumor-activated prodrugs. Over-expression of MGMT in hematopoietic stem cells to protect bone marrow from the toxic effects of chemotherapy is also a feasible selection. The future prospects and challenges of MGMT inhibitors in cancer chemotherapy were also discussed.

Autophagy protects bone marrow mesenchymal stem cells from palmitate‑induced apoptosis through the ROS‑JNK/p38 MAPK signaling pathways.

In recent years, the association between saturated fatty acids (FA) and bone cells has received a high level of attention. Previous studies have shown that palmitate (PA), a common saturated FA, can cause apoptosis in bone marrow mesenchymal stem cells (BMSCs). However, whether PA can induce autophagy, an important intracellular protection mechanism that is closely associated with apoptosis, in BMSCs is still unknown; the association between autophagy and apoptosis is also unclear. The aim of the present study was to determine whether PA can induce autophagy in BMSCs. When BMSCs were treated with PA for >18 h, p62 began to accumulate, indicating that autophagic flux was impaired by prolonged exposure to PA. In addition, the proportion of apoptotic cells was increased when autophagy was inhibited by the autophagy inhibitor 3-methyladenine. Furthermore, inducing autophagy by pretreating cells with rapamycin, a known inducer of autophagy, markedly reduced PA-induced apoptosis, suggesting that autophagy may serve a protective role in PA-induced apoptosis in BMSCs. PA also increased intracellular reactive oxygen species (ROS) production, which was decreased by the antioxidant N-Acetyl-cysteine, and promoted the activation of c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinase (MAPK). The addition of JNK and p38 MAPK inhibitors substantially reduced autophagy. Therefore, the results indicated that PA can induce autophagy in BMSCs and protect cells from PA-induced apoptosis through the ROS-JNK/p38 MAPK signaling pathways. These results may improve the general understanding of the mechanisms through which BMSCs adapt to PA-induced apoptosis. The present study also provides a novel approach for the prevention and treatment of PA-induced lipotoxicity.

A preliminary study of the protection of bone marrow in craniospinal irradiation

Objective To analyze the relationship between the dose volume of bone marrow and acute hematologic toxicity in craniospinal irradiation (CSI), and preliminarily explore the dosimetric differences in bone marrow between helical tomotherapy (HT) and volumetric modulated arc therapy (VMAT). Methods Clinical data and HT plans of twenty patients treated with CSI in the First Affiliated Hospital of Kunming Medical University were collected, and the HT plans were transferred back to the MIM system. The skull, mandible, clavicle, sternum, scapula, ribs, vertebrae and pelvis were successively delineated on the CT images and then the V2, V5, V10, V15, V20, V25, V30, V35, Dmax, Dmean of each aforementioned bone and total bone were read on DVH images. The correlation between those indexes and the incidence of ≥grade Ⅱ hematologic toxicity was subsequently analyzed. The images of 6 patients were selected and transferred to Monaco TPS and VMAT plans were completed. The dosimetric differences of those indexes were statistically compared between HT and VMAT. Statistical analyses were performed by using Spearman correlation analysis and Wilcoxon symbol rank-sum test. Results The incidence of ≥grade Ⅱ leukopenia was positively correlated with the V25 of pelvis and the V5 of total bones (P=0.038); the risk of ≥ grade Ⅱ thrombocytopenia was positive associated with the V20 of pelvis (P=0.041); the incidence of ≥ grade Ⅱ neutropenia was positively correlated with the V10 of vertebrae (P=0.036). There was no dosimetric difference of dose volume of vertebral and pelvis between HT and VMAT plans (P>0.05). Conclusions There is a positive correlation between ≥ grade Ⅱ leukopenia and the V25 of pelvis and the V5 of total bones. The V20 of pelvis shows a positive correlation with ≥ grade Ⅱ thrombocytopenia. A positive correlation is found between ≥ grade Ⅱ neutropenia and the V10 of vertebrae. The indices of vertebral and pelvis between HT and VMAT plans show no significant differences. Key words: Helical tomotherapy; Volumetric modulated arc therapy; Craniospinal irradiation; Bone marrow-sparing

The mechanism for the radioprotective effects of zymosan-A in mice.

It proved that Zymosan‐A protected the haematopoietic system from radiation‐induced damage via Toll‐Like Receptor2 in our previous study. In this study, we investigated the potential mechanism for the radioprotective effects of Zymosan‐A. The mice were treated with Zymosan‐A (50 mg/kg, dissolved in NS) via peritoneal injection 24 and 2 hours before ionizing radiation. Apoptosis of bone marrow cells and the levels of IL‐6, IL‐12, G‐CSF and GM‐CSF were evaluated by flow cytometry assay. DNA damage was determined by γ‐H2AX foci assay. In addition, RNA sequencing was performed to identify differentially expressed genes (DEGs). Zymosan‐A protected bone marrow cells from radiation‐induced apoptosis, up‐regulated IL‐6, IL‐12, G‐CSF and GM‐CSF in bone marrow cells. Zymosan‐A also protected cells from radiation‐induced DNA damage. Moreover, RNA sequencing analysis revealed that Zymosan‐A induced 131 DEGs involved in the regulation of immune system process and inflammatory response. The DEGs were mainly clustered in 18 KEGG pathways which were also associated with immune system processes. Zymosan‐A protected bone marrow cells from radiation‐induced apoptosis and up‐regulated IL‐6, IL‐12, G‐CSF and GM‐CSF. Moreover, Zymosan‐A might also exhibit radioprotective effects through regulating immune system process and inflammatory response. These results provided new knowledge regarding the radioprotective effect of Zymosan‐A.

Toll-like receptors and radiation protection.

Exposure to ionizing radiation (IR) causes severe injuries to the human body, and normal tissue toxicity also limits the further application of cancer radiotherapy. However, current clinically used radioprotective agents are difficult to produce satisfactory effects. Toll-Like receptor (TLR) is a kind of pattern recognition receptor (PRR) that has been extensively studied for radioprotection in recent years. Several TLR family members are closely related to radioprotection. In cultured cells, TLR2, TLR5 or TLR9 agonist was proved to inhibit radiation-induced apoptosis and increase cell survival. TLR5 ligand CBLB502 was reported to alleviate bone marrow and intestinal injuries in mice and rhesus monkeys. Activation of TLR4 by its agonist LPS can protect bone marrow damage and lower mice mortality after irradiation. TLR9 ligand also exhibited protective effects on mid jejunum. Moreover, some kinds of TLR agonists, such as TLR2/6 co-agonist CBLB613, were reported to be more effective in radioprotection than single TLR agonist. In conclusion, TLRs and their ligands provide novel strategies for radiation protection in nuclear accidents as well as protection of normal tissues during cancer radiotherapy.

Heme oxygenase-1 protects bone marrow mesenchymal stem cells from iron overload through decreasing reactive oxygen species and promoting IL-10 generation

Iron overload (IO) caused by frequent blood transfusion in hematological diseases has become a major concern. In this study, up-regulation of heme oxygenase-1 (HO-1), a protector against oxidative stress, was observed in bone marrow mesenchymal stem cells (BMMSCs) at the early stage of IO and had favorable prognosis in an IO mouse model. Given that the protective role of HO-1 in IO damage of BMMSCs was still unknown, the mechanism was explored in vitro and in vivo. BMMSCs were transfected with HO-1/siHO-1 in vitro, and the mouse model was established to further evaluate the effect of HO-1 on IO in vivo. As a result, HO-1 decreased the apoptotic rate of BMMSCs with IO through reducing intracellular reactive oxygen species (ROS) but increasing IL-10 secretion. In addition, IL-10 was mediated by HO-1 via the ERK pathway. Intracellular iron was down-regulated by hepcidin depending on IL-10. In conclusion, HO-1 protects BMMSCs from ROS by secreting IL-10 upon iron overload.

Angelica sinensis Polysaccharides Ameliorate Stress-Induced Premature Senescence of Hematopoietic Cell via Protecting Bone Marrow Stromal Cells from Oxidative Injuries Caused by 5-Fluorouracil.

Myelosuppression is the most common complication of chemotherapy. Decline of self-renewal capacity and stress-induced premature senescence (SIPS) of hematopoietic stem cells (HSCs) induced by chemotherapeutic agents may be the cause of long-term myelosuppression after chemotherapy. Whether the mechanism of SIPS of hematopoietic cells relates to chemotherapeutic injury occurred in hematopoietic microenvironment (HM) is still not well elucidated. This study explored the protective effect of Angelica sinensis polysaccharide (ASP), an acetone extract polysaccharide found as the major effective ingredients of a traditional Chinese medicinal herb named Chinese Angelica (Dong Quai), on oxidative damage of homo sapiens bone marrow/stroma cell line (HS-5) caused by 5-fluorouracil (5-FU), and the effect of ASP relieving oxidative stress in HM on SIPS of hematopoietic cells. Tumor-suppressive doses of 5-FU inhibited the growth of HS-5 in a dose-dependent and time-dependent manner. 5-FU induced HS-5 apoptosis and also accumulated cellular hallmarks of senescence including cell cycle arrest and typical senescence-associated β-galactosidase positive staining. The intracellular reactive oxygen species (ROS) was increased in 5-FU treated HS-5 cells and coinstantaneous with attenuated antioxidant capacity marked by superoxide dismutase and glutathione peroxidase. Oxidative stress initiated DNA damage indicated by increased γH2AX and 8-OHdG. Oxidative damage of HS-5 cells resulted in declined hematopoietic stimulating factors including stem cell factor (SCF), stromal cell-derived factor (SDF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), however, elevated inflammatory chemokines such as RANTES. In addition, gap junction channel protein expression and mediated intercellular communications were attenuated after 5-FU treatment. Significantly, co-culture on 5-FU treated HS-5 feeder layer resulted in less quantity of human umbilical cord blood-derived hematopoietic cells and CD34+ hematopoietic stem/progenitor cells (HSPCs), and SIPS of hematopoietic cells. However, it is noteworthy that ASP ameliorated SIPS of hematopoietic cells by the mechanism of protecting bone marrow stromal cells from chemotherapeutic injury via mitigating oxidative damage of stromal cells and improving their hematopoietic function. This study provides a new strategy to alleviate the complication of conventional cancer therapy using chemotherapeutic agents.

Protection of bone marrow, mononuclear, and CD34+ cells by enclosing within the biochemical compound solution during and after transplantation.

We have chosen collagen, chitosan acetate, hyaluronic acid, and propolis as model biochemical compound solution to determine the influence of cell carrier mechanics on cell viability and functionality during and after transplantation. Suspending of bone marrow (BM), mononuclear (MN), and CD34+ cells into a biochemical compounds solution is an attractive tool to achieve to protect and ensure reproducible deliver. Hyperglycemic rats were randomly divided into 2 groups: to receive no cell treatment or approximately 1×105 of BM, MN, and CD34+ cells within the PBS or biochemical compound solution. These cells were infused into the hyperglycemic rats on day 10 and again on day 20. At each time point, the animals were anaesthetized with ether, and 200μL of blood was drawn from the tail vein. Samples were collected to determine whether BM, MN, and CD34+ cell affected glucose content and insulin production. Our results exhibit the use of biochemical compound solution method to overcome the cell transplantation problem during and after injection of these cells into rats. These findings are supported by resulting in significantly greater insulin production and more decreased glucose content than cells injected in PBS only (P<0.05). These effects displayed the following hierarchy: hyaluronic acid>chitosan acetate>collagen>propolis solution. Our results showed that these compounds demonstrated a capacity to encapsulate the BM, MN, and CD34+ cells. It is proven by decreasing glucose content and increasing insulin secretion by pancreatic cells. The uniqueness of our study is the improvement of current transplantation efficiency.

Radiation protection and mitigation potential of phenylbutyrate: delivered via oral administration

Purpose: Phenylbutyrate (PB), a histone deacetylase inhibitor (HDACi) has demonstrated radiation protection in both in vitro and in vivo models. Studies previously demonstrated that PB and other HDAC inhibitors could inhibit radiation lethality in vivo by subcutaneous (s.c) injection. The objective of this study was to test the ability of oral PB treatment to protect against or to mitigate acute gamma radiation-induced lethality in vivo.Materials and methods: Human osteoblasts cells were used to evaluate radiation survival when PB was delivered pre- or post-radiation. A 30-day radiation lethality study was used to assess the radioprotective (pre-radiation) and radiomitigative (post-radiation) capability of PB. Possible mechanisms evaluated were antioxidant activity effects, HDAC inhibition, DNA damage, and hematological recovery.Results: Treatment of HOS cells with PB 50 μM either before or after radiation increased radiation resistance as assessed by clonogenic survival. Western blot studies showed that PB treatment acetylated histones in vivo and ameliorated the radiation-induced reduction in acetylated histone-4 (H4). Pre-radiation oral administration of PB (10 mg/kg) provided radioprotection against gamma radiation (7–11.5 Gy) with a dose reduction factor of 1.25 (p = 0.001). PB oral administration post-radiation provided moderate radiation mitigation against gamma radiation (7–11.5 Gy) and demonstrated a dose reduction factor of 1.18 (p = 0.05). PB pre-radiation and post-radiation treatment was associated with significant elevations in neutrophils and platelets and attenuation of DNA damage.Conclusions: These results indicate that oral PB has potential as a radiation protector and a radiation mitigator and that potential mechanisms of action include attenuation of DNA damage, antioxidant activity, and bone marrow protection.

Melatonin protects bone marrow mesenchymal stem cells against iron overload-induced aberrant differentiation and senescence.

Bone marrow mesenchymal stem cells (BMSCs) are an expandable population of stem cells which can differentiate into osteoblasts, chondrocytes and adipocytes. Dysfunction of BMSCs in response to pathological stimuli contributes to bone diseases. Melatonin, a hormone secreted from pineal gland, has been proved to be an important mediator in bone formation and mineralization. The aim of this study was to investigate whether melatonin protected against iron overload-induced dysfunction of BMSCs and its underlying mechanisms. Here, we found that iron overload induced by ferric ammonium citrate (FAC) caused irregularly morphological changes and markedly reduced the viability in BMSCs. Consistently, osteogenic differentiation of BMSCs was significantly inhibited by iron overload, but melatonin treatment rescued osteogenic differentiation of BMSCs. Furthermore, exposure to FAC led to the senescence in BMSCs, which was attenuated by melatonin as well. Meanwhile, melatonin was able to counter the reduction in cell proliferation by iron overload in BMSCs. In addition, protective effects of melatonin on iron overload-induced dysfunction of BMSCs were abolished by its inhibitor luzindole. Also, melatonin protected BMSCs against iron overload-induced ROS accumulation and membrane potential depolarization. Further study uncovered that melatonin inhibited the upregulation of p53, ERK and p38 protein expressions in BMSCs with iron overload. Collectively, melatonin plays a protective role in iron overload-induced osteogenic differentiation dysfunction and senescence through blocking ROS accumulation and p53/ERK/p38 activation.

Incorporation of Cerium Oxide into Hydroxyapatite Coating Protects Bone Marrow Stromal Cells Against H2O2-Induced Inhibition of Osteogenic Differentiation.

Oxidative stress exerts a key influence in osteoporosis in part by inhibiting osteogenic differentiation of bone marrow stromal cells (BMSCs). With their unique antioxidant properties and reported biocompatibility, cerium oxide (CeO2) ceramics exhibit promising potential for the treatment of osteoporosis resulting from oxidative stress. In this study, protective effects of CeO2-incorporated hydroxyapatite coatings (HA-10Ce and HA-30Ce) on the viability and osteogenic differentiation of H2O2-treated BMSCs were examined. CeO2-incorporated HA coatings enhanced cell viability and attenuated cell apoptosis caused by H2O2. An increase in CeO2 content in HA coatings better alleviated H2O2-induced inhibition of osteogenic differentiation by increasing alkaline phosphatase (ALP) activity, calcium deposition activity, and mRNA expression levels of osteogenesis markers runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OCN) in BMSCs. Furthermore, the H2O2-induced decrease of gene and protein expressions of β-catenin and cyclin D1 in the Wnt/β-catenin signaling pathway was successfully rescued by the CeO2 incorporated HA coatings. Besides, the decreased expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and the increased ratio of osteoprotegerin (OPG)/RANKL in BMSCs on the CeO2-modified coatings was observed, indicating the inhibition of osteoclastogenesis. The above results were mediated by the antioxidant properties of CeO2. The CeO2-incorporated HA coatings reversed the decreased superoxide dismutase (SOD) activity, reduced reactive oxygen species (ROS) generation, and suppressed the malondiadehyde (MDA) formation. The findings suggested that CeO2-modified HA coatings may be promising coating materials for osteoporotic bone regeneration.

Alternative Means of Estimating 131I Maximum Permissible Activity to Treat Thyroid Cancer.

To protect bone marrow from overirradiation, the maximum permissible activity (MPA) of 131I to treat thyroid cancer is that which limits the absorbed dose to blood (as a surrogate of marrow) to less than 200 cGy. The conventional approach (method 1) requires repeated γ-camera whole-body measurements along with blood samples. We sought to determine whether reliable MPA values can be obtained by simplified procedures. Methods: Data acquired over multiple time points were examined retrospectively for 65 thyroid cancer patients, referred to determine 131I uptake and MPA for initial treatment after thyroidectomy (n = 39), including 17 patients with compromised renal function and 22 patients with known (n = 16) or suspected (n = 6) metastases. The total absorbed dose to blood (DTotal) was the sum of mean whole-body γ-ray dose component (Dγ) from uncollimated γ-camera measurements and dose due to β emissions (Dβ) from blood samples. Method 2 estimated DTotal from Dβ alone, method 3 estimated DTotal from Dγ alone, and method 4 estimated DTotal from a single 48-h γ-camera measurement. MPA was computed as 200 cGy/DTotal for each DTotal estimate. Results: Method 2 had the strongest correlation with conventional method 1 (r = 0.98) and values similar to method 1 (21.0 ± 13.7 cGy/GBq vs. 21.0 ± 14.1 cGy/GBq, P = 0.11), whereas method 3 had a weaker (P = 0.001) correlation (r = 0.94) and method 4 had the weakest (P < 0.0001) correlation (r = 0.69) and lower dose (16.3 ± 14.8 cGy/GBq, P < 0.0001). Consequently, correlation with method 1 MPA was strongest for method 2 MPA (r = 0.99) and weakest for method 4 (r = 0. 75). Method 2 and method 1 values agreed equally well regardless of whether patients had been treated with 131I previously or had abnormal renal function. Conclusion: Because MPA based on blood measurements alone is comparable to MPA obtained with combined body counting and blood sampling, blood measurements alone are sufficient for determining MPA.

Abstract P2-11-09: A p53-based strategy for protecting normal breast tissue from chemotherapy-induced damage in breast conserving therapy

Abstract Introduction: Currently, the global rate of breast conserving therapy (BCT) increased steadily. Most patients with BCT are treated with chemotherapy. However, most chemotherapeutical drugs that kill cancer cells also cause undesirable injuries to normal breast tissues. Normal breast tissue damage will further cause destruction of female secondary sex characteristics, and ultimately affect the quality of patients' lives. Therefore, it is urgent to protect the normal breast tissues for BCT patients during chemotherapy. More and more evidence shows that chemotherapy-induced normal tissue damage is mainly caused by the activation of p53 pathway, which is separate from the tumor suppressor pathway of p53. Previous studies found that use of low-dose arsenic (LDA) could temporarily and reversibly suppresses p53 activation. There are recent studies showing LDA selectively protect bone marrow and gastrointestinal tract during cancer treatment. Therefore, we hypothesize that use of LDA to temporary inhibit p53 activity will be a new strategy to protect the breast normal tissues for breast conserving patients. Methods: Human breast epithelial cell line, MCF-10A, and three breast cancer cell lines MCF-7 (estrogen receptor and E6 expressed), MDA-MB-231 (triple negative and p53 mutated) and BT-474 (HER2 overexpressed and p53 mutated), were tested in this study. All cells were pretreated with either PBS or 100nM sodium arsenite for 12 hours, followed by 375 μM 5-fluorouracil (5-FU) or DMSO for 24, 48 and 72 hours. Cellular viability was determined by MTT assay and cell morphology was recorded under a light microscope. Results: Morphology changes after 5-FU treatment include: the cell density decreased, the cells became rounded in shape, the cell membrane atrophied, the cell nuclei underwent pyknosis, and the cells formed a globule with nuclear and cytoplasmic fragments surrounded by the cell membrane. LDA pretreated-MCF10A cells showed significant reduced growth inhibition by 5-FU at all detected time pointes as demonstrated by MTT assay and morphology observation. Interestingly, LDA treatment had negligible effect on survival in breast cancer cells. Table 1. Cell growth evaluated by MTT assay.Time after 5-FU treatment(h)Cell lineControl (%)LDA (%)5-FU (%)LDA+5-FU (%)24MCF-10A10099.3051.3878.28 MCF-710097.3268.5165.64 MDA-MB-23110094.9669.5669.13 BT-47410098.7971.5071.5948MCF-10A10097.3729.1347.74 MCF-710091.7649.9148.95 MDA-MB-23110093.769.048.81 BT-47410092.0460.4361.0072MCF-10A10096.3418.3443.64 MCF-710091.0142.0039.40 MDA-MB-23110093.596.445.39 BT-47410091.4635.7636.95 Conclusion: Temporary LDA pretreatment selectively protected normal tissue cells, but not tumor cells from toxicity of 5-FU chemotherapy. Our findings indicated that LDA pretreatment is a potential strategy to protect normal breast tissue during chemotherapy for BCT patients. RL and HS contributed equally to this work. Citation Format: Long M, Huang Y, Liu R, Liu R, Su H. A p53-based strategy for protecting normal breast tissue from chemotherapy-induced damage in breast conserving therapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-11-09.

Acupuncture Regulates the Notch Signaling Pathway in Bone Marrow Cells to Alleviate Cyclophosphamide-Induced Myelosuppression in S180 Tumor Bearing Mice

Objective: To evaluate the protein expression and distribution of Notch signaling-related, differentially expressed genes (DEGs) in the bone marrow cells of S180 tumor bearing mice. Additionally, verification of acupuncture regulated protein expression of these DEGs to protect bone marrow stem cells and alleviate cyclophosphamide (CTX) chemotherapy-induced myelosuppression was examined as well. Methods: Healthy male Kunming mice were inoculated with S180 sarcomas and further divided into blank control (group A), CTX group (group B), acupuncture (group C), and moxibustion (group D) groups. Animals in groups B, C, and D were intraperitoneally injected with single dose of 150 mg/kg CTX to generate the myelosuppressive model; while animals in group A were injected with an equivalent volume of sterile saline. After immobilizing the animals, mice in groups C and D received daily acupuncture and moxibustion, respectively at Dazhui, Geshu, Shenshu, and Zusanli acupoints; while mice in groups A and B received no daily treatment. Animals in each group were euthanized by cervical dislocation following five days of treatment and their femurs collected for marrow harvest. Small-throughput cDNA microarray was used for initial DEG screening, followed by immunohistochemistry, real-time quantitative PCR (RT-qPCR), and Western Blot analysis to detect protein expression and quantity of the Notch signaling-related DEGs in the bone marrow cells. Results: Expression of numb proteins (i.e., numb1 and numb2) was found to be up regulated while notch2 and jag1 were down regulated in bone marrow cells of acupuncture treated, tumor bearing mice. Conclusion: Acupuncture regulated the changes of Notch signaling-related DEGs in mouse bone marrow cells. The data indicates that acupuncture treatment may alleviate the CTX chemotherapy-induced myelosuppression associated with tumor burden, suggesting that might be the key mechanism to improve bone marrow function.

Chemotherapy Efficacy on Human Breast Cancer Xenograft in Mice Remains Effective in Combination with Myelo001, a Novel Orally Bioavailable Small Molecule to Treat Myelosuppression

IntroductionGranulocyte colony-stimulating factor (G-CSF) and its biosimilars remain the only approved therapy for chemotherapy-induced neutropenia to date (Smith TJ, Bohlke K, Lyman GH, et al. Recommendations for the Use of WBC Growth Factors: American Society of Clinical Oncology Clinical Practice Guideline Update. J Clin Oncol. 2015;33(28):3199-212).Despite the available treatment, patients who experience febrile neutropenia still suffered from a higher risk of mortality (Lyman GH, Michels SL, Reynolds MW, Barron R, Tomic KS, Yu J. Risk of mortality in patients with cancer who experience febrile neutropenia. Cancer. 2010;116:5555-5563).Previously we have shown that imidazolyl ethanamide pentandioic acid (IEPA, Myelo001) consistently reduces chemotherapy-induced leukopenia during cyclophosphamide (CP) treatment (Pleimes D, Flechsig S, Meyer M. Effect of IEPA, a Novel Orally Bioavailable Small Molecule, on Chemotherapy-Induced Myelosuppression. Poster presented at American Society of Hematology Annual Meeting and Exposition. December 8, 2014. San Francisco, CA).Prior non-clinical and clinical studies suggested that the mode of action of Myelo001 is the protection of the hematopoietic bone marrow against the cytotoxic effects of chemotherapy (CTX). A related question is whether Myelo001 also inhibits the effects of CTX on malignant tissue. Here we present the results of the first in vivo study that has investigated whether the effects of CTX on a human breast cancer xenograft in mice remains effective in combination with Myelo001.MethodsFemale athymic nude mice (Crl:NU(NCr)-Foxn1nu) were injected with MDA-MB-231 into the flank. Once the average tumor size reached 100 to 150 mm3, animals were sorted into 6 groups, as shown in Fig. 1, and treatment started. Assessed parameters were: body weight and caliper measurements of tumor size, observation of treatment-related side effects, mortality, and complete blood count (CBC) analysis of blood from mandibular bleeds. The tumor volume was calculated using the following formula:ResultsThe mean tumor volume of the negative control and Myelo001 group increased exponentially and significantly over the study period (Fig. 1). Both groups treated with CP displayed a tumor growth delay (TGD) until about day 20, after which exponential growth occurred. Treatment with Doxorubicin (Doxo) displayed the same TGD as the CP groups. Furthermore, Doxo was able to reduce the tumor volume consistently throughout the study.Figure 1: Mean tumor volume for each treatment group, plotted with 95% confidence interval and treatment design of the xenograft studyThe study endpoint for individual animals was defined as reaching a tumor volume > 1500 mm3 or day 47 of the study, whichever occurred first. No significant difference was observed when comparing the negative control and Myelo001 groups (Fig. 2). Concomitant treatment with Myelo001 together with either CP or Doxo also did not show significant differences compared to the respective group without Myelo001 treatment.Figure 2: Box-and-whisker plot displaying the time to endpoint for each treatment group and response summary of the presented studyThe CBC analysis showed that the median white blood cell and neutrophil counts were significantly smaller for the CP and Doxo groups without Myelo001 on study days 22, 33, and 47 (endpoint) compared to the control group. In combination with Myelo001, the white blood cell count during Doxo treatment displayed a higher trend. The median neutrophil count during treatment with CP and Myelo001 was significantly higher at the endpoint compared to the treatment without Myelo001. (CBC data not shown within the abstract)ConclusionsThis study shows that treatment of human breast cancer xenografts in nude mice with either CP or Doxo was not influenced by concomitant treatment with Myelo001. Thus, the tumor is not protected from CTX when treating leukopenia with Myelo001. The CBC analysis displayed advantages of Myelo001, retaining higher numbers of white blood cells and neutrophils during CTX, but had limitations due to low frequency measurement points and alternating assessment of animals to avoid an influence on the main study objective. In combination with the previous report of the protective properties of Myelo001, this drug is clearly a promising development candidate for chemotherapy-induced myelosuppression. [Display omitted] [Display omitted] [Display omitted] DisclosuresBrehm:Myelo Therapeutics GmbH: Employment. Donnimath:Myelo Therapeutics GmbH: Employment. Pleimes:Myelo Therapeutics GmbH: Employment, Equity Ownership; CC-Ery GmbH: Consultancy, Honoraria; Bayer: Consultancy, Equity Ownership, Honoraria.