Significant Increase In Necrosis Research Articles

Ellagic Acid Triggers the Necrosis of Differentiated Human Enterocytes Exposed to 3-Nitro-Tyrosine: An MS-Based Proteomic Study.

To study the molecular basis of the toxicological effect of a dietary nitrosated amino acid, namely, 3-nitrotyrosine (3-NT), differentiated human enterocytes were exposed to dietary concentrations of this species (200 μM) and analyzed for flow cytometry, protein oxidation markers and MS-based proteomics. The possible protective role of a dietary phytochemical, ellagic acid (EA) (200 μM), was also tested. The results revealed that cell viability was significantly affected by exposure to 3-NT, with a concomitant significant increase in necrosis (p < 0.05). 3-NT affected several biological processes, such as histocompatibility complex class II (MHC class II), and pathways related to type 3 metabotropic glutamate receptors binding. Addition of EA to 3-NT-treated cells stimulated the toxicological effects of the latter by reducing the abundance of proteins involved in mitochondrial conformation. These results emphasize the impact of dietary nitrosated amino acids in intestinal cell physiology and warn about the potential negative effects of ellagic acid when combined with noxious metabolites.

Large-Volume Fat Grafting: Identifying Risk Factors for Fat Necrosis.

Fat necrosis is a well-recognized complication following autologous fat grafting. The purpose of this study was to evaluate the incidence of fat necrosis after large-volume fat grafting and identify risk factors for fat necrosis. A retrospective review was performed on 83 consecutive patients who underwent large-volume fat grafting (>100 cc) to the breast performed by the senior author (L.P.B.) between September of 2011 and May of 2016. Fat necrosis was defined as palpable nodules, or nodules seen on imaging. A total of 148 breasts underwent 170 autologous fat transplantations. Indications included the following: 72 reconstructions after surgical therapy and 98 cosmetic augmentations. Mean age was 48 years, median graft volume was 300 cc, and median length of follow-up 423 days. Overall incidence of necrosis was 32.9 percent, with 47.8 percent in previously irradiated patients. Increased incidence of necrosis was associated with increasing fat graft volumes (OR, 1.002; p = 0.032), increasing body mass index (OR, 1.13; p = 0.04), and simultaneous implant exchange with fat ( p = 0.003). Fat grafting volumes greater than 450 cc in a single breast were also associated with an increase in fat necrosis ( p = 0.04). Within a group of six patients who had bilateral fat grafting with unilateral radiation therapy, there was a significant increase in necrosis on the irradiated side ( p = 0.015). In a cohort of non-BRAVA patients, reconstruction (compared to augmentation) was associated with fat necrosis ( p = 0.039). Increased rates of fat necrosis were associated with volumes greater than 450 cc, patients undergoing concurrent implant exchange with fat grafting, and fat grafting after a history of lumpectomy or mastectomy without preexpansion. In addition, radiation therapy may be associated with a higher rate of complications. Risk, III.

Facile synthesis and anticancer activity of novel dihydropyrimidinone derivatives

Abstract The enaminone, (2E)-3-(dimethylamino)-1-(3,4,5-trimethoxyphenyl) prop-2-en-1-one was prepared by refluxing 3,4,5-trimethoxy acetophenone with dimethylformamide dimethylacetal (DMF–DMA) without solvent for 12 h. The dihydropyrimidinone derivatives (1–9) were prepared by reacting enaminone, substituted benzaldehydes and urea in glacial acetic acid. The compounds (1–9) were synthesized in significant yield using one step multicomponent reaction. Structures of all the novel synthesized compounds were characterized and confirmed by various spectroscopic methods. The compounds were evaluated for their anti-cancer activity against HepG2 cancer cell line. Compound 9 displayed significant anti-cancer activity. During the apoptotic assay, it showed a significant increase in necrosis from 1.97% to 12.18% as compared to the control. Mechanism of anti-proliferation was performed by cell cycle distribution assay, which showed a decrease in G2+M from 12.90 to 8.13 as compared to control.

Histological features of masticatory muscles after botulinum toxin A injection into the right masseter muscle of dystrophin deficient (mdx-) mice

Objective/backgroundThe most frequently used animal model for human DMD (Duchenne muscular dystrophy) research is the mdx mouse. In both species, characteristic histological changes like inflammation, muscle fiber degeneration and fibrosis are the same, but in contrast to humans, in mdx mice, phases of muscle fiber degeneration are compensated by regeneration processes. AimTherefore, the interest of this study was to evaluate histological features in masticatory muscles after BTX-A injection into the right masseter muscle of wild type and dystrophic (mdx) mice, illustrating de- and regeneration processes induced by this substance. Material and methodsThe right masseter muscle of 100 days old healthy and mdx mice were selectively paralyzed by a single intramuscular BTX-A injection. Masseter as well as temporal muscle of injection and non-injection side were carefully dissected 21 days and 42 days after injection, respectively, and fiber diameter, cell nuclei position, necrosis and collagen content were analyzed histomorphologically in order to evaluate de- and regeneration processes in these muscles. Statistical analysis was performed using SigmaStat Software and Mann Whitney U-test (significance level: p < 0.05). ResultsAt both investigation periods and in both mouse strains fiber diameter was significantly reduced and collagen content was significantly increased in the right injected masseter muscle whereas fiber diameters in mdx mice were much smaller, and these differences were even more apparent at the second investigation period. Necrosis and central located nuclei could generally be found in all mdx mice muscles investigated with an amount of centronucleation exceeding 60%, and a significant increase of necrosis six weeks after injection. In wild type mice central located nuclei could primarily be found in the treated masseter muscle with a portion of 2.7%, and this portion decreased after six weeks, whereas in mdx mice a decrease could also be seen in the non-injected muscles. In contrast, in wild type mice necrosis was not apparent at any time and in all muscles investigated. ConclusionFrom our results it can be concluded that in mdx mice masticatory muscles de- and regeneration processes were extended, triggered by a selective BTX-A injection, or mdx mice at this age, independently of BTX-A treatment, went through another cycle of de- and regeneration as a characteristic of this disease.

Pharmacological inhibition of mTORC1 increases CCKBR-specific tumor uptake of radiolabeled minigastrin analogue [177Lu]Lu-PP-F11N.

Rationale: A high tumor-to-healthy-tissue uptake ratio of radiolabeled ligands is an essential prerequisite for safe and effective peptide receptor radionuclide therapy (PRRT). In the present study, we searched for novel opportunities to increase tumor-specific uptake of the radiolabeled minigastrin analogue [177Lu]Lu-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 ([177Lu]Lu-PP-F11N), that targets the cholecystokinin B receptor (CCKBR) in human cancers.Methods: A kinase inhibitor library screen followed by proliferation and internalization assays were employed to identify compounds which can increase uptake of [177Lu]Lu-PP-F11N in CCKBR-transfected human epidermoid carcinoma A431 cells and natural CCKBR-expressing rat pancreatic acinar AR42J cells. Western blot (WB) analysis verified the inhibition of the signaling pathways and the CCKBR level, whereas the cell-based assay analyzed arrestin recruitment. Biodistribution and SPECT imaging of the A431/CCKBR xenograft mouse model as well as histological analysis of the dissected tumors were used for in vivo validation.Results: Our screen identified the inhibitors of mammalian target of rapamycin complex 1 (mTORC1), which increased cell uptake of [177Lu]Lu-PP-F11N. Pharmacological mTORC1 inhibition by RAD001 and metformin increased internalization of [177Lu]Lu-PP-F11N in A431/CCKBR and in AR42J cells. Analysis of protein lysates from RAD001-treated cells revealed increased levels of CCKBR (2.2-fold) and inhibition of S6 phosphorylation. PP-F11N induced recruitment of β-arrestin1/2 and ERK1/2 phosphorylation. In A431/CCKBR-tumor bearing nude mice, 3 or 5 days of RAD001 pretreatment significantly enhanced tumor-specific uptake of [177Lu]Lu-PP-F11N (ratio [RAD001/Control] of 1.56 or 1.79, respectively), whereas metformin treatment did not show a significant difference. Quantification of SPECT/CT images confirmed higher uptake of [177Lu]Lu-PP-F11N in RAD001-treated tumors with ratios [RAD001/Control] of average and maximum concentration reaching 3.11 and 3.17, respectively. HE staining and IHC of RAD001-treated tumors showed a significant increase in necrosis (1.4% control vs.10.6% of necrotic area) and the reduction of proliferative (80% control vs. 61% of Ki67 positive cells) and mitotically active cells (1.08% control vs. 0.75% of mitotic figures). No significant difference in the tumor vascularization was observed after five-day RAD001 or metformin treatment.Conclusions: Our data demonstrates, that increased CCKBR protein level by RAD001 pretreatment has the potential to improve tumor uptake of [177Lu]Lu-PP-F11N and provides proof-of-concept for the development of molecular strategies aimed at enhancing the level of the targeted receptor, to increase the efficacy of PRRT and nuclear imaging.

Cytotoxic Action of N-aryl, Furan-derived Aminophosphonates against HT29 and HCT116 Cancer Cell Lines

The anticancer activity of aminophosphonic derivatives has been described extensively, some recent papers included furan-derived aminophosphonates and their cytostatic action against various cancer cells. A series of twelve furan-derived dibenzyl and diphenyl aminophosphonates 2a-f and 3a-f was synthesized and tested in aspect of their cytotoxic action on two cell lines of colorectal cancer: HT29 and HCT116. Seven of them are new compounds, while the rest five have already been published by us, together with their cytotoxic action against squamous esophageal cancer cells. To estimate the cytotoxicity effect of tested compounds MTT test was used. Pro-apoptotic activity of five selected compounds was evaluated using APC Annexin V Apoptosis Detection Kit on a flow cytometer. Quantification of caspases 3/7 activity was performed using Caspase-Glo® 3/7 Assay Kit. Five of these aminophosphonates showed significant cytotoxicity higher than those of cisplatin. Simultaneous evaluation of their cytotoxicity against PBLs revealed that these compounds are rather not harmful for regular human lymphocytes. Tests on apoptosis vs. their necrotic actions on cells were performed with selected compounds showing the most significant cytotoxicity against cancer cells and all tested compounds did not induce significant increase of necrosis in cells, whereas they showed moderate-to-strong proapoptotic actions even at the lowest applied concentration. Caspase 3/7 activity results confirmed proapoptotic properties of tested aminophosphonates. From among studied compounds, dibenzyl N-phenyl substituted amino(2-furyl)methylphsophonates were found to be more potent compounds in aspect of their antiproliferative action than the corresponding diphenyl derivatives.

Abstract: Identifying Risk Factors for Fat Necrosis After Autologous Fat Transplantation to the Breast

PURPOSE: Fat necrosis is a concerning complication after autologous fat grafting for breast reconstruction and aesthetic augmentation. It can present as a palpable mass concerning for malignancy. As such, we sought to evaluate risk factors for fat necrosis and identify methods for prevention. METHODS: We reviewed 83 consecutive patients who underwent large volume fat transplantation to the breast performed by the senior author between September 2011-May 2015. Patients with a minimum of 100cc of transplanted fat were included. RESULTS: A total of 148 breasts underwent 170 autologous fat transplantations. Indications included 72 reconstructions after surgical therapy for breast cancer and 98 cosmetic augmentations. BRAVA was used in 133 cases- 45 reconstructions and 88 augmentations. Twenty-three cases had previous radiation therapy. Seventeen breasts had simultaneous implant exchange with fat (SIEF). Mean age was 48 years, with average graft volume of 336.9cc (100 - 1000), and average length of follow-up 485 days (14 - 1219). Overall incidence of necrosis was 32.9%, with 47.8% in irradiated patients compared to 30.6% in nonirradiated patients. Increased rates of necrosis were demonstrated in groups with fat grating volumes over 500cc (p=0.001) and those who underwent SIEF(p=0.003). Within a group of six patients that had bilateral fat grafting with unilateral radiation, there was a significant increase in necrosis on the radiated side (p=0.015). In a cohort of non-BRAVA patients, reconstruction (compared to augmentation) was associated with necrosis (p=0.036). CONCLUSION: In this study, increased rates of fat necrosis were associated with volumes greater than 500cc, SIEF, radiation, and non-BRAVA reconstruction.

Abstract 3213: Hypomethylation therapy leads to immune polarization and improved efficacy of immunotherapy in transgenic model of pancreatic cancer

Abstract BACKGROUND Despite significant activity of immunotherapies in several malignancies, their efficacy in pancreatic cancer has been limited. One possible explanation may be the absence of a tumor microenvironment and immune cells that are necessary. We have demonstrated that 5-aza-2-deoxycitidine (DAC) had a profound effect on pancreatic cancer associated stroma and up-regulated many immune pathways. Therefore we proposed that hypomethylating therapy may alter the microenvironment to allow immue checkpoint inhibitors greater therapeutic efficacy. METHODS Transgenic mice that spontaneously develop pancreatic cancer (Kras, Trp53, Pdx-1 Cre [KPC]) underwent weekly transabdominal ultrasound to detect mice initially with larger (6-8 mm) and subsequently with smaller (3-5 mm) tumors. PBS or DAC (5 mg/kg) and anti -PD1H or isotype control antibody were administered every 72 hours intraperitoneally for 3 doses. Tumor size was monitored biweekly and 3D tumor volume reconstruction was used to estimate size. The immune cell infiltrate was characterized by quantitative immunohistochemistry or by FACS. RESULTS Mice treated with DAC with 6-8 mm tumors demonstrated a significantly increased survival and tumor necrosis as well as polarization of the infiltrating macrophages towards an M1 (CD86, CD163) phenotype and a trend towards an increase in infiltrating CD8 cells compared with controls. The shift in the T cell population and the macrophage phenotypes was not seen in the spleen or peripheral blood of either wild type or tumor bearing animals treated with DAC compared with controls. We subsequently treated mice with 3 doses of DAC followed by 3 doses of anti -PD1H and compared them to anti-PD1H alone in KPC mice with smaller (3-5 mm) tumors. In DAC + anti-PD1H we noted a decrease in the growth rate of tumors, an increased survival and a significant increase in necrosis and CD8 infiltrates, as well as a significant decrease in Ki67 and increase in Tunel staining compared with anti-PD1H alone. CONCLUSIONS Our results suggest that DAC pre-treatment prior to checkpoint inhibitors may favorably polarize the tumor infiltrating immune cell population, lead to increased recruitment of CD8 positive T cells, result in marked tumor necrosis and slow tumor growth. These effects ultimately contribute to increased therapeutic efficacy of checkpoint inhibitors in pancreatic cancer. We plan to optimize the efficacy of epigenetic therapy and checkpoint inhibitors by using other monoclonal antibodies (anti-PD1, PD1L)and different dosing regimens. Citation Format: Tamas Gonda, Jarwei Fang, Catheine Do, Anna Zhukovskaya, Martha Salas, Benjamin Tycko. Hypomethylation therapy leads to immune polarization and improved efficacy of immunotherapy in transgenic model of pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3213.

Abstract 491: Tumor response to cabozantinib in the TH-MYCN GEM model of neuroblastoma

Abstract Neuroblastoma (NB) is the most common extra-cranial solid tumor in childhood. The proto-oncogene MYCN is amplified in 25% of NB, and is associated with enhanced angiogenesis and poor survival. Vascular endothelial growth factor (VEGF) is the most potent angiogenic growth factor and, with the hepatocyte growth factor (HGF)/c-MET signalling pathway, is implicated in the progression of human NB. Cabozantinib (CBZ) is a small-molecule kinase inhibitor with potent activity against VEGFR2 and MET, currently in clinical trials against NB. In this study, the efficacy of CBZ was evaluated in tumors spontaneously arising in the TH-MYCN GEM model of high-risk NB, previously shown to faithfully recapitulate the radiological and hypervascular characteristics of childhood disease. TH-MYCN mice with spontaneous abdominal tumors were treated daily with either vehicle or 30mg/kg CBZ. Non-invasive T2-weighted anatomical MRI revealed that CBZ induced significant (p&amp;lt;0.001) tumor growth arrest after 48 hours compared to control. Quantitative functional MRI showed this volumetric response was associated with significant decreases in both the native longitudinal MRI relaxation time T1 (p&amp;lt;0.01) a measure of the ratio of bound to free water in tissue reflecting the increased extracellular space in tumors, and transverse MRI relaxation rate R2* (p&amp;lt;0.05), sensitive to paramagnetic deoxyhemoglobin. Histopathological analysis revealed a significant increase in necrosis (p&amp;lt;0.01), and significant decrease in microvessel density (p&amp;lt;0.01) and VEGFR2 scoring (p&amp;lt;0.02) in CBZ-treated tumors compared to control. Significant (p&amp;lt;0.01) tumor growth delay was maintained after 7 days treatment with CBZ compared to control, which translated into a significant improvement in survival rate (p&amp;lt;0.0001). This study demonstrates that CBZ is efficacious against, and conveys a survival benefit for the TH-MYCN GEM model of NB. The data reinforces native T1 as a biomarker of response to anti-vascular therapy due to its sensitivity to therapy-induced damage to erythrocytes and/or cell death-mediated increase in macromolecules into the extracellular space. Relatively fast R2* rates in NB arising in the TH-MYCN model are strongly associated with vascular instability, characterised by numerous regions of aggregated paramagnetic erythrocytes (“blood lakes”), and treatment-induced thrombosis with the pan-VEGFR inhibitor cediranib which increased tumor R2*. The CBZ-induced decrease in R2* is clearly associated with vascular disruption, hence precluding the extravasation of deoxygenated red blood cells. Citation Format: Gilberto S. Almeida, Philippa King, Yann Jamin, Albert Hallsworth, Hannah Webber, Sergey Popov, Andrew D.J. Pearson, Louis Chesler, Simon P. Robinson. Tumor response to cabozantinib in the TH-MYCN GEM model of neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 491. doi:10.1158/1538-7445.AM2015-491

Abstract 2484: IR700-conjugated anti-CD44 monoclonal antibody for phototherapy theranostics of triple-negative breast cancer

Abstract CD44 transmembrane glycoproteins are cell adhesion molecules that play a role in cancer cell proliferation, migration, and invasion.1 CD44 expression is associated with stem-like breast cancer cells1 and is upregulated under hypoxia2 making it a significant target. Monoclonal antibody (mAb)-linked to photosensitizers have achieved targeted phototherapy.3 We developed a CD44 mAb-targeted photosensitizer complex for combined fluorescent detection and phototherapy (PT) of CD44 expressing triple negative breast cancer cells. The mAb conjugate is activated by near-infrared (NIR) light, and demonstrates phototoxic effects selectively in CD44 expressing breast cancer cells following cell membrane binding. CD44 mAb was conjugated with a NIR phthalocyanine dye, IR700, to form CD44-IR700. MB-MDA-231 (CD44 positive), MCF-7 and BT-474 (CD44 negative) were selected for the cell studies. SDS-PAGE, UV spectroscopy and confocal fluorescence imaging were performed to determine purity, composition and cellular localization of the conjugate. Microscopic observation and cell viability tests were performed to study CD44-specific cell death. Tumor models were established by inoculating 2×106 MB-MDA-231 or BT-474 cells in the upper-right mammary fat pad of athymic female mice. In vivo fluorescence imaging was performed on a Li-Cor Pearl® Impulse scanner. Tumor bearing mice (n = 5 per group) injected with PBS, IR700, anti-CD44 mAb, IgG-IR700 and CD44-IR700 were monitored over a 3 week-period and sacrificed to harvest tumors. NIR light at a dose of 30 J/cm2 was applied on day 1 after injection. CD44-IR700 demonstrated preferential binding to the cell membrane of MB-MDA-231 but not MCF-7 or BT-474 cells. CD44-specific cell death was initiated by CD44-IR700-mediated PT (&amp;gt;90% of MB-MDA-231 cells were killed immediately, while 88% of MCF-7 cells and 90% of BT-474 cells were alive). Irradiation itself was harmless to cells and there was no cytotoxicity associated with IR700 and CD44 antibody or with CD44-IR700 in the absence of irradiation. In vivo fluorescence imaging verified the 3-fold increased retention of CD44-IR700 in MB-MDA-231 tumors compared to BT-474 tumors. CD44-IR700 injection together with a therapeutic dose of NIR light exposure resulted in significant growth delay in MB-MDA-231 tumors, but not BT-474 tumors. We did not observe tumor growth delay in separate groups of mice injected with PBS, IR700, anti-CD44 mAb, or IgG-IR700, irrespective of NIR light application. Histological studies confirmed a significant increase of necrosis in the treated MDA-MB-231 tumors. This novel PT theranostic strategy provides a promising opportunity for treating CD44 expressing primary and metastatic triple negative breast cancer.

RhoA Gtpase Regulates Erythroblast Cytokinesis and Enucleation By Dynamic Coordination Of The Microtubule-Actomyosin Machineries For Successful Abscission

RhoA GTPase is known to regulate cell adhesion, actomyosin cytoskeleton, and cytokinesis. Specifically, it contributes to cytokinesis in sea urchin embryos, Xenopus and HeLa cells by associating with the microtubule ends at the abscission area marking the position for the daughter cell separation and by activating myosin through phosphorylation of the myosin regulatory light chain (MRLC) (Piekny et al, Trends in Cell Biology 2005). Erythroblast enucleation, the process through which orthochromatic erythroblasts expel their nucleus during the final stage of mammalian erythropoiesis, is a multi-step process resembling asymmetric cytokinesis (reviewed by Li, Developmental Cell 2013). It requires establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring between reticulocyte and pyrenocyte (Konstantinidis et al, Blood 2012).To define the mechanistic contribution of RhoA signaling in terminal erythroid maturation, we generated mice with erythroid specific RhoA deletion (EpoR-GFPcre-driven). Erythroid-specific deletion of RhoA, confirmed in the CD71-positive cells by PCR and immunoblotting, caused severe anemia, fatal in utero by E15.5. RhoA-deficient peripheral primitive blood cells were large with significant poikilocytosis and anisocytosis, frequently binuclear or multinuclear with polyploidy of the genetic material as demonstrated by flow cytometry with propidium iodide, and with incomplete clearance of the Golgi network and mitochondria. RhoAΔ/Δ fetal livers had progressively decreased cellularity. The erythroblast populations as determined by CD71-Ter119-FSC flow cytometry as well as by multispectral high-speed cell imaging in flow, demonstrated progressive decline and significantly decreased reticulocyte production (n=3, p<0.05), compatible with a fatal intrauterine anemia. RhoAΔ/Δ definitive erythroblasts also exhibited increased frequency of polyploidy. These polyploid cells were Ki67-negative by immunohistochemistry, indicating that they were not actively mitotic. Apoptosis was not significantly increased in the RhoA-deficient erythroblasts as determined by Annexin-V positivity. However, the late RhoAΔ/Δ erythroblasts demonstrated a significant increase in necrosis as determined by 7AAD and Annexin-V double-positivity (n=3, p<0.05). Increased phosphorylation of p53 was evident by immunoblotting, likely functioning as a polyploidy checkpoint after failure of abscission due to RhoA deficiency. Further evidence that RhoA is significant for microtubule organization in erythroblasts was attained by immuno-localization of RhoA at the microtubule ends and by decreased nuclear polarization in EpoR-Cre RhoAΔ/Δ orthochromatic erythroblasts, as shown by decreased delta centroid Ter119-Draq5 (the distance between the centers of the erythroblast and the nucleus) in multispectral high-speed cell imaging in flow. Phosphorylated myosin regulatory light chain (pMRLC) was found significantly decreased by immunoblotting of RhoAΔ/Δ fetal liver erythroblasts, indicating that RhoA controls phosphorylation of MRLC in erythropoiesis. Increased PAK phosphorylation was noted pointing towards compensatory upregulation of Rac GTPases in the RhoA-deficient cells, which was confirmed by quantitative RT-PCR. We further examined the erythroblastic islands in RhoAΔ/Δfetal liver by transmission electron microscopy and found that RhoA-deficient erythroblasts, frequently dysplastic and binucleated, were loosely associated with the central macrophage and there was a paucity of intracellular vacuoles in comparison to WT erythroblasts, indicating possible defects in vesicular transport, which has also been shown to play a role in erythroblast enucleation (Keerthivasan et al, Blood 2010).Thus, RhoA GTPase regulates erythroblast differentiation and enucleation by dynamic coordination of the microtubule-actomyosin machineries to achieve successful abscission. In addition, RhoA may also regulate erythroblast adhesive interactions with the central macrophage in erythroblastic islands and intracellular vesicular transport. Our results reveal novel molecular components and cellular processes in erythropoiesis that may be important for improving the efficiency of red blood cell production in vitro. Disclosures:No relevant conflicts of interest to declare.

Acute tumour response to the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142886) evaluated by non-invasive diffusion-weighted MRI

Background:Non-invasive imaging biomarkers underpin the development of molecularly targeted anti-cancer drugs. This study evaluates tumour apparent diffusion coefficient (ADC), measured by diffusion-weighted magnetic resonance imaging (DW-MRI), as a biomarker of response to the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142886) in human tumour xenografts.Methods:Nude mice bearing human BRAFV600D WM266.4 melanoma or BRAFV600E Colo205 colon carcinoma xenografts were treated for 4 days with vehicle or selumetinib. DW-MRI was performed before and 2 h after the last dose and excised tumours analysed for levels of phospho-ERK1/2, cleaved caspase 3 (CC3) and necrosis.Results:Selumetinib treatment induced tumour stasis and reduced ERK1/2 phosphorylation in both WM266.4 and Colo205 tumour xenografts. Relative to day 0, mean tumour ADC was unchanged in the control groups but was significantly increased by up to 1.6-fold in selumetinib-treated WM266.4 and Colo205 tumours. Histological analysis revealed a significant increase in necrosis in selumetinib-treated WM266.4 and Colo205 xenografts and CC3 staining in selumetinib-treated Colo205 tumours relative to controls.Conclusion:Changes in ADC following treatment with the MEK1/2 inhibitor selumetinib in responsive human tumour xenografts were concomitant with induction of tumour cell death. ADC may provide a useful non-invasive pharmacodynamic biomarker for early clinical assessment of response to selumetinib and other MEK-ERK1/2 signalling-targeted therapies.

Abstract 551: The Dual Effect of Cigarette Smoke on Macrophage Abca1-dependent Cholesterol Efflux and Protease Production Leads to Decreased Collagen and Increased Necrosis in Plaques of ApoE Deficient Mice

Epidemiological evidence links smoking to an increase in the incidence and severity of cardiovascular disease. However, the molecular mechanisms responsible for susceptibility are unknown. The accumulation of lipids in macrophages induces an inflammatory response, with secretion of cytokines and proteases, and leads to necrosis and loss of collagen content within atherosclerotic lesions. Therefore, the effect of cigarette smoke on lipids in the atherosclerotic process and consequences was examined. Initial studies revealed that cigarette smoke attenuated cholesterol efflux. Subsequently, the end result of cigarette smoke exposure on atherosclerotic lesions in ApoE deficient mice demonstrated a significant increase in necrosis along with a dramatic decrease in the collagen content of lesions in ApoE deficient mice exposed to smoke for 20 weeks. Cigarette smoke extract (CSE) impaired the cholesterol efflux ability for ApoAI and correlated with ABCA1 loss and increased MMPs (9, 12, 13, in human THP-1 cells additionally MMP1 is increased), TLR4/Myd88 and TNFα expression in mouse bone marrow derived macrophages (BMdM) in vitro and in vivo in peritoneal macrophages elicited after 5 days of smoke exposure. Furthermore, by increasing ABCA1 expression with LXR agonist treatment the CSE-induced up regulation of MMPs (MMP-9 and 13) and TNFα was inhibited signifying a correlation between the cigarette smoke induced loss of ABC transporters and MMP induction. Moreover, BMDMs isolated from Cre-LysM-ABCA1 deficient mice displayed an increase in mRNA expression of MMPs (9, 12, 13) at a basal level, which was appreciably augmented with CSE as compared to control macrophages. The LXR induced decrease in MMPs and TNFα expression as well as decreased MMP-9 activity in macrophages was confirmed to be ABCA1-dependent since ABCA1 KO macrophages exposed to CSE did not exhibit this response. These findings establish that the effect of cigarette smoke on macrophage ABCA1-dependent cholesterol efflux impairment leads to both an increase in proteases and inflammatory cytokines and this combination of events is likely synergistic in ultimately generating a more vulnerable plaque through the increase in plaque necrosis and a decrease in collagen content.

Polyazamacrocycles as Potential Antitumor Agents for Human Prostate Cancer Cells

Polyazamacrocycles are currently being studied and used in a variety of applications beyond their traditional place in supramolecular and co-ordination chemistry. This study suggests additional applications of these compounds with particular emphasis on their use as antiproliferative agents that could be potentially used to treat cancer. Four polyazamacrocycles were tested in human prostate cancer LNCaP and prostate epithelial PNTA1 cells to analyze changes in cell proliferation and cell death capabilities. Their intracellular localization was also evaluated by confocal microscopy. The results show a decrease in proliferation rate and cell viability of LNCaP and PNTA1, after treatment with these compounds. The decrease in the number of viable cells is similar for the majority of the compounds studied, and at higher concentration, the proliferation efficiency decreased significantly in the cell lines studied. Also, our results suggest that L and L2 induce early apoptosis in PNTA1 cells and late apoptosis/necrosis in LNCaP cells. The compounds did not induce a significant increase in necrosis of both cell types. Although the compounds did not localize in a unique organelle, all of them have as main target the Golgi apparatus and other localization profiles differed depending on the cell line.

Development of a Mouse Model of Abdominal Cutaneous Flaps for Breast Reconstruction

Autologous tissue transfer, in addition to replacing tissue that was lost during injury or surgery, offers women an excellent option to improve cosmetic appearance and self-confidence following mastectomy due to breast cancer. However, flap necrosis is a complication in obese patients undergoing this procedure. We created a mouse model to study the flap-related complications that leads to decreased flap survival in autologous breast reconstruction.MethodsLeft superficial inferior epigastric (SIE) pedicle abdominal-cutaneous flaps were elevated in 8 week-old, obese ob/ob male mice and their lean littermates. Flaps were followed by serial photography. Area of flap necrosis was measured at 7 days. Statistical analysis was performed.ResultsNecrosis was observed at the distal margin of the flaps, in both lean and obese groups. Lean left SIE flaps (n = 8) had a total area flap necrosis of 9.1% at 7 days whereas obese left SIE flaps (n = 8) had a total area flap necrosis of 45.5% at 7 days. Obese flaps had a statistically significant increase in necrosis compared to the lean flaps, p = 0.001.ConclusionsThere was a significant difference between flap survival in lean and obese SIE pedicle flaps in our mouse model. We have developed the first flap model of obesity utilizing the superficial epigastric pedicle in the mouse. This model is optimal for future studies to dissect out mechanisms that lead to the complications related to flap survival for breast reconstruction, especially in obese subjects.

Effects of cold atmospheric plasma on mucosal tissue culture

Thermal plasmas have been commonly used in medical applications such as plasma ablation and blood coagulation. Newer developments show that plasmas can be generated with ion temperatures close to room temperature: these non-thermal or so-called cold atmospheric plasmas (CAPs) therefore open up a wide range of further biomedical applications. Based on the understanding of the bactericidal, virucidal and fungicidal properties of CAPs, information about the effects of CAP on mucosal cells and tissue is still lacking. Therefore this study focuses on the interaction of CAP with healthy head and neck mucosal cells on a molecular level.To analyse this interaction in detail, fresh tissue samples from healthy nasal and pharyngeal mucosa were harvested during surgery, assembled to a three-dimensional tissue culture model (mini organ cultures) and treated with CAP for different treatment times. Effects on the viability, necrosis induction and mutagenic activity were evaluated with the trypan blue exclusion test, Annexin-V/PI staining and alkaline microgel electrophoresis (comet assay). Trypan blue exclusion test revealed that the CAP treatment significantly decreases the cell viability for all tested treatment times (5, 10, 30, 60 and 120 s; p < 0.05), but only a treatment time of 120 s showed a cytotoxic effect as the viability dropped below 90%. Annexin-V/PI staining revealed a significant increase in necrosis in CAP treated pharyngeal tissue cultures for treatment times of 60 and 120 s (p < 0.05). For nasal tissue this effect was already detected for a 30 s treatment (p < 0.05). Comet assay analysis showed no mutagenic effects after exposure to CAP.

Elevated Activation of ERK1 and ERK2 Accompany Enhanced Liver Injury Following Alcohol Binge in Chronically Ethanol‐Fed Rats

Binge drinking after chronic ethanol consumption is one of the important factors contributing to the progression of steatosis to steatohepatitis. The molecular mechanisms of this effect remain poorly understood. We have therefore examined in rats the effect of single and repeat ethanol binge superimposed on chronic ethanol intake on liver injury, activation of mitogen-activated protein kinases (MAPKs), and gene expression. Rats were chronically treated with ethanol in liquid diet for 4 weeks followed by single ethanol binge (5 gm/kg body weight) or 3 similar repeated doses of ethanol. Serum alcohol and alanine amino transferase (ALT) levels were determined by enzymatic methods. Steatosis was assessed by histology and hepatic triglycerides. Activation of MAPK, 90S ribosomal kinase (RSK), and caspase 3 were evaluated by Western blot. Levels of mRNA for tumor necrosis factor alpha (TNFα), early growth response-1 (egr-1), and plasminogen activator inhibitor-1 (PAI-1) were measured by real-time qRT-PCR. Chronic ethanol treatment resulted in mild steatosis and necrosis, whereas chronic ethanol followed by binge group exhibited marked steatosis and significant increase in necrosis. Chronic binge group also showed significant increase (compared with chronic ethanol alone) in the phosphorylation of extracellular regulated kinase 1 (ERK1), ERK2, and RSK. Phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK did not increase by the binge. Ethanol binge, after chronic ethanol intake, caused increase in mRNA for egr-1 and PAI-1, but not TNFα. Chronic ethanol exposure increases the susceptibility of rat liver to increased injury by 1 or 3 repeat binge. Among other alterations, the activated levels of ERK1, and more so ERK2, were remarkably amplified by binge suggesting a role of these isotypes in the binge amplification of the injury. In contrast, p38 MAPK and JNK1/2 activities were not amplified. These binge-induced changes were also reflected in the increases in the RNA levels for egr-1 and PAI-1. This study offers chronic followed by repeat binge as a model for the study of progression of liver injury by ethanol and highlights the involvement of ERK1 and ERK2 isotypes in the amplification of liver injury by binge ethanol.

Electrochemical treatment: An investigation of dose-response relationships using an isolated liver perfusion model

Background/Aim:Ablative techniques such as radiofrequency ablation or non-thermal electrochemical treatment (ECT) are used to manage unresectable liver metastases. Although ECT is not affected by the cooling effect from adjacent vessels, there is a paucity of data available on ECT.Materials and Methods:We used porcine livers to establish an organ model with portal venous and hepatic arterial blood flow for a standardized analysis of the relationship between dose (electric charge) and response (volume of necrosis).Results:This model allowed us to study pressure-controlled perfusion of portal venous and hepatic arterial circulation in the absence of a capillary leak. A specially designed guiding template helped us place platinum electrodes at reproducible locations. With two electrodes, there was a linear relationship between charges of no more than 200 C and necrosis. The relationship was logarithmic at charges of 400-600 C. Larger electrode spacing led to a significant increase in necrosis. We measured pH values of 0.9 (range: 0.6-1.3) at the anode and 12.6 (range: 11.6-13.4) at the cathode.Conclusions:Using a perfusion model, we established an experimental design that allowed us to study ECT in the liver of large animals without experiments on living animals. An electrode template helped us improve the standardized analysis of dose-response relationships. ECT created reproducible and sharply demarcated areas of necrosis, the size of which depended on the charge delivered as well as on the number and spacing of electrodes. Doses higher than 600 C require longer treatment times but do not increase the area of necrosis (logarithmic dose-response relationship).

Repeated Doses of Adriamycin Infused In Swine through the Intraosseous Route In the Same Bone May Result In Osteomyelitis

AbstractAbstract 4343 Introduction:Intraosseous (IO) vascular access has been shown to be a safe and effective alternative to central venous lines for the use of emergency drugs, but its utility for the delivery of chemotherapy has not been determined. A 2007 study detected marginal changes in bone morphology after delivery of single doses of a variety of vesicant chemotherapeutic agents. However, Adriamycin in the previous study involved only a single dose and examined only acute effects to bone marrow morphology. This study was designed to determine the safety of multiple rounds of Adriamycin delivery via the IO route repeatedly in the same bone in a swine model. Methods:An IO needle (EZ IO, Vidacare Corporation, Shavano Park, TX) was aseptically placed into both tibias of 14 Yorkshire swine. On 3 occasions, 21 days apart, a dose of Adriamycin (60mg/m2) was delivered over 15 minutes followed by 2 minutes of saline infusion through a proximal tibia. For control purposes, the contra-lateral tibia received saline infusions. A radiologist reviewed films pre and post drug delivery. Bone samples were harvested 30 days post last drug delivery. Paired t-tests were used to compare changes between treated and saline controls. Two pathologists separately reviewed the slides of 14 animals. One pathologist scored the slides and both pathologists' comments were recorded in a masked fashion without knowledge of treatment groups, according to animal number and left or right limb. A one-way analysis of variance was used to evaluate the treatment effect (Adriamycin vs. saline control). A two-sided alpha level of 0.05 was used to define statistical significance. Results:After the 2nd infusion, visual examination revealed small dime-sized lesions over the injection site in 4 animals. After the third injection almost all animals developed some form of leg swelling and limping. Radiologically, osteomyelitis was detected in 6 animals (43%), 2 of which had fractures to the proximal head of the tibia. An additional 4 animals had fractures to the proximal head of the tibia without osteomyelitis. Of the 10 affected animals, half the complications occurred after the second Adriamycin dose and half occurred after the third dose. Histologically, there was no statistical significance for the parameters of marrow cell loss (p=0.85), bone spicule loss (p=1.0), blood in the field (p=0.20), or necrosis (p=0.33) between drug infused limbs and their paired controls. Matrix formation and scar formation were significantly greater in the drug limbs when compared to the saline control limbs (p<0.001 and p<0.05, respectively). There was no statistical significance for the parameters of hypocellularity (p=0.77), marrow degeneration (p=0.33), or blood (p=0.33) comparing drug limb to control limb. Severity of necrosis (p= 0.001) and fibrosis (p=0.001) formation were significantly higher for the drug limb compared to the control limb. Summary and Conclusions:Results suggest the repeated doses of Adriamycin in the same bone led to formation of osteomyelitis in 6 of 14. Two of the 6 swine had fracture confirmed at study end; 4 other pigs fracture were without signs of osteomyelitis while there was no evidence of osteomyelitis in saline-infused bones. The Adriamycin infusion in the same bone caused a significant increase in matrix and scar formation in histological samples taken from the drug limb compared to the saline control limb. In addition, there was a significant increase in necrosis and fibrosis in the drug infused limb. On the long-term evaluation, these increases suggest that Adriamycin has some direct effect on inducing bone pathology. Additionally, these complications may have been exacerbated by additional factors—including hurricane evacuation. A number of recommendations have been given to address these issues. The most important considerations could be the alternation of infusion limbs during drug delivery and increased post flush volumes. Disclosures:Hoskins:Vidacare Corporation: Research Funding. Philbeck:Vidacare Corporation: Employment. Miller:Vidacare Corporation: Employment, Equity Ownership. Kramer:Vidacare Corporation: Consultancy, Patents & Royalties, Research Funding.

Abstract 4465: In vivo antitumor characterization of the hypoxia-activated prodrug TH-302 in a preclinical SCLC xenograft model

Abstract The hypoxia activated prodrug TH-302, has showed anti-tumor activity in a variety of preclinical in vivo models. In hypoxic conditions, TH-302 acts as a DNA crosslinking agent by the reductase- and oxygen concentration-dependent release of bromo-IPM. In the present study, we investigated the anti-cancer mechanisms of TH-302 in a small cell lung cancer (SCLC) xenograft model with a focus on selective effects on the hypoxic compartment and tumor vasculature. The ectopic xenograft model was set up by s.c. implantation of 5×106 H82 human SCLC cells into the flank of nude mice. When tumor size reached 500 to 700mm3, animals were randomized (5-8 mice/group) and were injected i.p. with a single dose of 150mg/kg TH-302 or vehicle. Tumors were harvested for frozen sections 24, 48 and 72 hours after TH-302 treatment. Pimonidazole, for detection of hypoxia, was administered at 60mg/kg i.p. 1 hour before sacrifice, and to assess the effect of TH-302 on tumor vascular perfusion, Hoechst 33342 was injected at 10mg/kg i.v. into the tail vein 1 min before sacrifice. Anti-CD31 antibody was utilized as a marker for the microvasculature. Morphometric analysis was conducted by point counting and with Image-Pro Plus 6.0 software. Previous studies showed that TH-302 as a single agent, exhibited efficacy in the H82 xenograft model with tumor growth inhibition at about 55%. In the current study, anti-tumor activity was evaluated by assessment of tumor necrosis after H&amp;E staining. The necrotic fraction in the vehicle treated tumor was 7.3% and reached 17.2%, 37.9% and 34.8% after 24, 48 and 72hrs of TH-302 treatment, respectively. A significant increase of necrosis occurred from 48hrs after the treatment (p&amp;lt;0.05). The pimonidazole-positive hypoxic fraction decreased after TH-302 treatment and reached a significant difference at 48hrs (6.3% ± 1.2% in vehicle vs. 1.8% ± 1.1% in the 48hr treatment group, p&amp;lt;0.05). Tumor vessel density and vascular perfusion in the non-necrotic area, as characterized by CD31-positive microvasculature cells and Hoechst 33342-positive cells, showed no significant difference between vehicle and any of the treated groups. There was significant correlation between tumor vessel density (CD31) and tumor vessel flow (Hoechst 33342) (R2=0.28, p&amp;lt;0.05). The results demonstrate an antitumor effect of TH-302 through cytotoxic activity on the hypoxic compartment in the tumor. In addition, a bystander effect is likely, given the observation that the necrosis fraction was 38% 48hrs after TH-302 treatment while the hypoxic fraction was less than 8%. Unlike tirapazamine, another hypoxia-activated prodrug which can cause vascular dysfunction, TH-302 appears to exhibit minor effects on the vasculature. The results support an in vivo tumor hypoxia targeting mechanism of TH-302. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4465.