Radiation Biological Toximetry Using Circulating Cell-Free DNA (cfDNA) for Rapid Radiation/Nuclear Triage.

Optimal triage biodosimetry would include risk stratification within minutes, and it would provide useful triage despite heterogeneous dosimetry, cytokine therapy, mixed radiation quality, race, and age. For regulatory approval, the U.S. Food and Drug Administration (FDA) Biodosimetry Guidance requires suitability for purpose and a validated species-independent mechanism. Circulating cell-free DNA (cfDNA) concentration assays may provide such triage information. To test this hypothesis, cfDNA concentrations were measured in unprocessed monkey plasma using a branched DNA (bDNA) technique with a laboratory developed test. The cfDNA levels, along with hematopoietic parameters, were measured over a 7-day period in Rhesus macaques receiving total body radiation doses ranging from 1 to 6.5 Gy. Low-dose irradiation (0-2 Gy) was easily distinguished from high-dose whole-body exposures (5.5 and 6.5 Gy). Fold changes in cfDNA in the monkey model were comparable to those measured in a bone marrow transplant patient receiving a supralethal radiation dose, suggesting that the lethal threshold of cfDNA concentrations may be similar across species. Average cfDNA levels were 50 ± 40 ng/mL [±1 standard deviation (SD)] pre-irradiation, 120 ± 13 ng/mL at 1 Gy; 242 ± 71 ng/mL at 2 Gy; 607 ± 54 at 5.5 Gy; and 1585 ± 351 at 6.5 Gy (±1 SD). There was an exponential increase in cfDNA concentration with radiation dose. Comparison of the monkey model with the mouse model and the Guskova model, developed using Chernobyl responder data, further demonstrated correlation across species, supporting a similar mechanism of action. The test is available commercially in a Clinical Laboratory Improvement Amendments (CLIA) ready form in the U.S. and the European Union. The remaining challenges include developing methods for further simplification of specimen processing and assay evaluation, as well as more accurate calibration of the triage category with cfDNA concentration cutoffs.

Durability of and role of AKT in FGF7p urothelial protection against cyclophosphamide.

We previously identified a peptide derived from human fibroblast growth factor 7 (FGF7p) that blocks urothelial apoptosis similar to full-length FGF7, although effects of FGF7p on urothelial repair are unknown. Also, while urothelial AKT activation downstream of FGF7p correlated with the anti-apoptotic effects, we have not directly interrogated the role of AKT in mediating the cytoprotection. Our goal was to assess effects of FGF7p on urothelial repair and the role of AKT signaling in mediating the cytoprotective effects of FGF7p. We performed hematoxylin and eosin (H&E), TUNEL, and/or immunofluorescence (IF) staining for various markers in FGF7p-treated mice 28 days after giving cyclophosphamide or after co-administering a systemic AKT antagonist with FGF7p 24 h after cyclophosphamide. Vehicle-treated and injured mice had hyperplastic urothelium, incomplete return of mature superficial cell markers, ongoing proliferation, and continued presence of basal progenitor markers 28 days after injury; conversely, FGF7p-treated mice had normal numbers of urothelial cell layers, nearly complete return of superficial cell markers, limited proliferation and fewer basal progenitor cells 28 days post-injury. Vehicle-treated mice also had ectopic lumenal basal progenitor cell markers, while FGF7p had none 28 days after cyclophosphamide. Co-administration of an AKT inhibitor largely abrogated FGF7p-driven AKT activation and cytoprotection in urothelium 24 h after injury. Thus, FGF7p drives faster and higher fidelity urothelial repair by limiting apoptotic injury via AKT signaling, similar to full-length FGF7. Finally, FGF7p is much less expensive to synthesize and has a longer shelf life and higher purity than FGF7.

FGF7 peptide (FGF7p) mimetic mitigates bladder urothelial injury from cyclophosphamide.

Although full-length fibroblast growth factor 7 (FGF7) blocks cyclophosphamide-induced urothelial apoptosis in mice, limitations include high production costs because of its large size. We previously identified a small peptide derived from FGF2 that mitigated acute radiation syndrome as well as full-length FGF2. Based on the sequence of the FGF2 peptide, we synthesized a corresponding 19 amino acid FGF7 peptide (FGF7p). Our objectives were to determine if systemic FGF7p triggered the downstream targets and protected against cyclophosphamide bladder injury similar to full-length FGF7. We administered FGF7p or vehicle subcutaneously (SQ) to mice subjected to no injury or intraperitoneal (IP) cyclophosphamide and harvested bladders 1 day after injury. We then performed hematoxylin and eosin, TUNEL and immunofluorescence (IF) staining. In uninjured mice, a 20 mg/kg threshold FGF7p dose induced expression of phosphorylated (activated) FRS2α (pFRS2α), and pAKT in urothelium (consistent with cytoprotective effects of FGF7). We then gave FGF7p (20 mg/kg) or vehicle at 72 and 48 h prior to cyclophosphamide. One day after injury, TUNEL staining revealed many more apoptotic urothelial cells with vehicle treatment versus FGF7p treatment. IF for pAKT and readouts of two anti-apoptotic AKT targets (BAD and mTORC1) revealed minimal staining with vehicle treatment, but strong urothelial expression for all markers with FGF7p treatment. In conclusion, FGF7p appears to block bladder urothelial apoptosis via AKT and its targets, similar to FGF7. FGF7p is much more inexpensive to make and has a longer shelf life and higher purity than FGF7.

Normal tissue and tumor microenvironment adaptations to aerobic exercise enhance doxorubicin anti-tumor efficacy and ameliorate its cardiotoxicity in retired breeder mice.

Aerobic exercise is receiving increased recognition in oncology for its multiple purported benefits. Exercise is known to induce physiologic adaptations that improve patient quality-of-life parameters as well as all-cause mortality. There also is a growing body of evidence that exercise may directly alter the tumor microenvironment to influence tumor growth, metastasis, and response to anticancer therapies. Furthermore, the physiologic adaptations to exercise in normal tissues may protect against treatment-associated toxicity and allow for greater treatment tolerance. However, the exercise prescription required to induce these beneficial tumor-related outcomes remains unclear. This study characterized the aerobic adaptations to voluntary wheel running in normal tissues and the tumor microenvironment. Female, retired breeder BALB/c mice and syngeneic breast adenocarcinoma cells were utilized in primary tumor and metastasis models. Aerobic exercise was found to induce numerous adaptations across various tissues in these mice, although primary tumor growth and metastasis were largely unaffected. However, intratumoral hypoxia and global metabolism were altered in the tumors of exercising hosts relative to non-wheel running controls. Doxorubicin chemotherapy also was found to be more efficacious at delaying tumor growth with adjuvant aerobic exercise. Additionally, doxorubicin-induced cardiac toxicity was ameliorated in exercising hosts relative to non-wheel running controls. Taken together, these data suggest that the normal tissue and tumor microenvironment adaptations to aerobic exercise can improve doxorubicin efficacy while simultaneously limiting its toxicity.

Reliability of blood lactate as a measure of exercise intensity in different strains of mice during forced treadmill running.

Exercise has long been known to be beneficial to human health. Studies aimed at understanding the effects of exercise specifically focus on predetermined exercise intensities defined by measuring the aerobic capacity of each individual. Many disease models involving animal training often establish aerobic capacity by using the maximal lactate steady state (MLSS), a widely used method in humans that has frequently been used in rodent studies. The MLSS is defined as the highest exercise intensity at which blood lactate concentration remains constant and is roughly equivalent to 70-80% of maximal aerobic capacity. Due to our up-coming experiments investigating the effect of different exercise intensities in specific strains of tumor-bearing mice, the aim of the present study was to determine the MLSS in athymic nude (NCr nu/nu and NMRI), CDF1, and C3H mice by treadmill running at increasing speeds. However, despite thorough exercise acclimation and the use of different exercise protocols and aversive stimuli, less than half of the experiments across strains pointed towards an established MLSS. Moreover, gently prodding the mice during low to moderate intensity running caused a 30-121% (p<0.05) increase in blood lactate concentration compared to running without stimulation, further questioning the use of lactate as a measure of exercise intensity. Overall, MLSS is difficult to determine and large variations of blood lactate levels were observed depending on the exercise protocol, mice handling strategy and strain. This should be considered when planning experiments in mice using forced exercise protocols.

Stability and Mobility of Magnetic Nanoparticles in Biological Environments Determined from Dynamic Magnetic Susceptibility Measurements.

Low tumor accumulation following systemic delivery remains a key challenge for advancing many cancer nanomedicines. One obstacle in engineering nanoparticles for high tumor accumulation is a lack of techniques to monitor their stability and mobility in situ. One way to monitor the stability and mobility of magnetic nanoparticles biological fluids in situ is through dynamic magnetic susceptibility measurements (DMS), which under certain conditions provide a measure of the particle's rotational diffusivity. For magnetic nanoparticles modified to have commonly used biomedical surface coatings, we describe a systematic comparison of DMS measurements in whole blood and tumor tissue explants. DMS measurements clearly demonstrated that stability and mobility changed over time and from one medium to another for each different coating. It was found that nanoparticles coated with covalently grafted, dense layers of PEG were the only ones to show good stability and mobility in all settings tested. These studies illustrate the utility of DMS measurements to estimate the stability and mobility of nanoparticles in situ, and which can provide insights that lead to engineering better nanoparticles for in vivo use.

Cathepsin L inactivation leads to multimodal inhibition of prostate cancer cell dissemination in a preclinical bone metastasis model.

It is estimated that approximately 90% of patients with advanced prostate cancer develop bone metastases; an occurrence that results in a substantial reduction in the quality of life and a drastic worsening of prognosis. The development of novel therapeutic strategies that impair the metastatic process and associated skeletal adversities is therefore critical to improving prostate cancer patient survival. Recognition of the importance of Cathepsin L (CTSL) to metastatic dissemination of cancer cells has led to the development of several CTSL inhibition strategies. The present investigation employed intra-cardiac injection of human PC-3ML prostate cancer cells into nude mice to examine tumor cell dissemination in a preclinical bone metastasis model. CTSL knockdown confirmed the validity of targeting this protease and subsequent intervention studies with the small molecule CTSL inhibitor KGP94 resulted in a significant reduction in metastatic tumor burden in the bone and an improvement in overall survival. CTSL inhibition by KGP94 also led to a significant impairment of tumor initiated angiogenesis. Furthermore, KGP94 treatment decreased osteoclast formation and bone resorptive function, thus, perturbing the reciprocal interactions between tumor cells and osteoclasts within the bone microenvironment which typically result in bone loss and aggressive growth of metastases. These functional effects were accompanied by a significant downregulation of NFκB signaling activity and expression of osteoclastogenesis related NFκB target genes. Collectively, these data indicate that the CTSL inhibitor KGP94 has the potential to alleviate metastatic disease progression and associated skeletal morbidities and hence may have utility in the treatment of advanced prostate cancer patients.

MRI-based computational model of heterogeneous tracer transport following local infusion into a mouse hind limb tumor.

Systemic drug delivery to solid tumors involving macromolecular therapeutic agents is challenging for many reasons. Amongst them is their chaotic microvasculature which often leads to inadequate and uneven uptake of the drug. Localized drug delivery can circumvent such obstacles and convection-enhanced delivery (CED)--controlled infusion of the drug directly into the tissue--has emerged as a promising delivery method for distributing macromolecules over larger tissue volumes. In this study, a three-dimensional MR image-based computational porous media transport model accounting for realistic anatomical geometry and tumor leakiness was developed for predicting the interstitial flow field and distribution of albumin tracer following CED into the hind-limb tumor (KHT sarcoma) in a mouse. Sensitivity of the model to changes in infusion flow rate, catheter placement and tissue hydraulic conductivity were investigated. The model predictions suggest that 1) tracer distribution is asymmetric due to heterogeneous porosity; 2) tracer distribution volume varies linearly with infusion volume within the whole leg, and exponentially within the tumor reaching a maximum steady-state value; 3) infusion at the center of the tumor with high flow rates leads to maximum tracer coverage in the tumor with minimal leakage outside; and 4) increasing the tissue hydraulic conductivity lowers the tumor interstitial fluid pressure and decreases the tracer distribution volume within the whole leg and tumor. The model thus predicts that the interstitial fluid flow and drug transport is sensitive to porosity and changes in extracellular space. This image-based model thus serves as a potential tool for exploring the effects of transport heterogeneity in tumors.

Transcriptional repression of VEGF by ZNF24: mechanistic studies and vascular consequences in vivo.

VEGF is a key regulator of normal and pathologic angiogenesis. Although many trans-activating factors of VEGF have been described, the transcriptional repression of VEGF remains much less understood. We have previously reported the identification of a SCAN domain-containing C2H2 zinc finger protein, ZNF24, that represses the transcription of VEGF. In the present study, we identify the mechanism by which ZNF24 represses VEGF transcription. Using reporter gene and electrophoretic mobility shift assays, we identify an 11-bp fragment of the proximal VEGF promoter as the ZNF24-binding site that is essential for ZNF24-mediated repression. We demonstrate in 2 in vivo models the potent inhibitory effect of ZNF24 on the vasculature. Expression of human ZNF24 induced in vivo vascular defects consistent with those induced by VEGF knockdown using a transgenic zebrafish model. These defects could be rescued by VEGF overexpression. Overexpression of ZNF24 in human breast cancer cells also inhibited tumor angiogenesis in an in vivo tumor model. Analyses of human breast cancer tissues showed that ZNF24 and VEGF levels were inversely correlated in malignant compared with normal tissues. These data demonstrate that ZNF24 represses VEGF transcription through direct binding to an 11-bp fragment of the VEGF proximal promoter and that it functions as a negative regulator of tumor growth by inhibiting angiogenesis.

Impact of the SRC inhibitor dasatinib on the metastatic phenotype of human prostate cancer cells.

SRC, a non-receptor tyrosine kinase, is frequently over-expressed and highly activated in blood as well as solid tumors in various organs, including prostate, and has been associated with aggressive disease and a poor patient prognosis. Prostate cancer patients with a high risk of developing metastases have few treatment options, none of which can result in a durable cure. Therefore, the aim of the present study was to examine the impact of a SRC inhibitor, dasatinib, on the ability of human prostate cancer cell to complete key steps in the metastatic process, including invasion and angiogenesis. Dasatinib treatment impaired the metastatic phenotypes of the human prostate cancer cell lines, PC-3, DU-145, and LNCaP, by significantly reducing migration and invasion in modified Boyden chambers. Inhibition of phosphorylation, and therefore enhanced activation, of SRC and key downstream signaling pathway elements, including FAK, STAT3, Paxillin, and Akt, as determined by Western blotting, also was observed. This suggests that dasatinib interferes with critical cell functions associated with the metastatic cascade. Dasatinib also had direct effects on the ability of microvascular endothelial cells to form tubes in vitro and impaired the ability of PC-3 cells to induce angiogenesis in vivo. In conclusion, the present findings suggest that SRC inhibition by dasatinib may have utility in reducing the metastatic spread of prostate cancer cells.

Support of a free radical mechanism for enhanced antitumor efficacy of the microtubule disruptor OXi4503.

Unlike normal blood vessels, the unique characteristics of an expanding, disorganized and leaky tumor vascular network can be targeted for therapeutic gain by vascular disrupting agents (VDAs), which promote rapid and selective collapse of tumor vessels, causing extensive secondary cancer cell death. A hallmark observation following VDA treatment is the survival of neoplastic cells at the tumor periphery. However, comparative studies with the second generation tubulin-binding VDA OXi4503 indicate that the viable rim of tumor tissue remaining following treatment with this agent is significantly smaller than that seen for the lead VDA, combretastatin. OXi4503 is the cis-isomer of CA1P and it has been speculated that this agent's increased antitumor efficacy may be due to its reported metabolism to orthoquinone intermediates leading to the formation of cytotoxic free radicals. To examine this possibility in situ, KHT sarcoma-bearing mice were treated with either the cis- or trans-isomer of CA1P. Since both isomers can form quinone intermediates but only the cis-isomer binds tubulin, such a comparison allows the effects of vascular collapse to be evaluated independently from those caused by the reactive hydroxyl groups. The results showed that the cis-isomer (OXi4503) significantly impaired tumor blood flow leading to secondary tumor cell death and >95% tumor necrosis 24h post drug exposure. Treatment with the trans-isomer had no effect on these parameters. However, the combination of the trans-isomer with combretastatin increased the antitumor efficacy of the latter agent to near that of OXi4503. These findings indicate that while the predominant in vivo effect of OXi4503 is clearly due to microtubule collapse and vascular shut-down, the formation of toxic free radicals likely contributes to its enhanced potency.

Impact of the Src inhibitor saracatinib on the metastatic phenotype of a fibrosarcoma (KHT) tumor model.

BACKGROUND: Src, a non-receptor tyrosine kinase frequently overexpressed and highly activated in malignancies, has been associated with a poor patient prognosis. The aim of the present studies was to examine the impact of an Src inhibitor (saracatinib) on a highly metastatic murine sarcoma cell line (KHT). MATERIALS AND METHODS: Phosphorylation of Src and downstream effectors was determined using Western immunoblotting. Cell cycle was analyzed by flow cytometry using propidium iodide DNA staining, migration and invasion in modified Boyden chambers, activated MMP-9 by gel zymography, and visualization of pSrc and pFAK by confocal immunofluorescence. The number of KHT lung nodules in saracatinib-treated mice was compared to controls. RESULTS: Saracatinib inhibited major pathways in the metastatic cascade in vitro, including Src and FAK activation. Functions required for metastasis, such as migration and invasion, were reduced when cells were exposed to 0.5 µM and 1.0 µM saracatinib, respectively (p<0.0001). Pretreatment of KHT cells with either 1 µM or 5 µM saracatinib prior to tail vein injection decreased lung colonies in mice from 13.0 to 5.0 (p<0.05) and less than 1.0 (p<0.01), respectively. CONCLUSION: These findings suggest that Src inhibition by saracatinib may reduce the metastatic activity of tumor cells.

Development of information regarding susceptibility to heat illness using the cooperative extension agency model in Kentucky.

CONTEXT: Dissemination of information regarding the latest research findings in rehabilitative health care is often limited to professional journals. OBJECTIVE: The purpose of the paper is to describe opportunities to better distribute scientific information to wider swaths than normally contained within a readership of a journal, to describe a process to deliver important information via the Cooperative Extension Service, and provide an example of such an informational brochure. DESIGN: An interdisciplinary approach was developed to provide access to a larger cohort of individuals the latest research findings regarding heat and hydration. DATA EXTRACTION: CINAHL, Medline, and Sport Discus were reviewed from 1966 to 2006 using the terms Heat, Hydration, Rhabdomyolysis, Rehabilitation, Heat Exhaustion, Heat Stroke, and Dehydration. DATA SYNTHESIS: We found substantial information describing recommendations for preventing, recognizing, and treating illness due to variance in heat and hydration. The information was succinctly summarized, converted to a 7th grade reading level, and shared with a larger audience via a unique model available through Cooperative Extension Agencies. CONCLUSION: Providing scientific information via a Cooperative Extension Model enables sharing of information from experts to communities. This methodology increases the distribution of the latest scientific knowledge to broader audiences.

Soy isoflavones exert differential effects on androgen responsive genes in LNCaP human prostate cancer cells.

The high consumption of soy isoflavones in Asian diets has been correlated to a lower incidence of clinically important cases of prostate cancer. This study characterized the effects of a soy-derived isoflavone concentrate (ISF) on growth and gene expression profiles in the LNCaP, an androgen-sensitive human prostate cancer cell line. ISF caused a dose-dependent decrease in viability (P < 0.05) and DNA synthesis (P < 0.01), as well as an accumulation of cells in G(2)/M, and G(0)/G(1) phases of the cell cycle compared with controls. Using Affymetrix oligonucleotide DNA microarrays (U133A), we determined that ISF upregulated 80 genes and downregulated 33 genes (P < 0.05) involving androgen-regulated genes and pathways controlling cell cycle, metabolism, and intracellular trafficking. Changes in the expression of the genes of interest, identified by microarrays, were validated by Western immunoblot, Northern blot, and luciferase reporter assays. Prostate-specific antigen, homeobox protein NKX3, and cyclin B mRNA were significantly reduced, whereas mRNA was significantly upregulated for p21(CIP1), a major cell cycle inhibitory protein, and fatty acid and cholesterol synthesis pathway genes. ISF also significantly increased cyclin-dependent kinase inhibitor p27(KIP1) and FOXO3A/FKHRL1, a forkhead transcription factor. A differential pattern of androgen-regulated genes was apparent with genes involved in prostate cancer progression being downregulated by ISF, whereas metabolism genes were upregulated. In summary, we found that ISF inhibits the growth of LNCaP cells through the modulation of cell cycle progression and the differential expression of androgen-regulated genes. Thus, ISF treatment serves to identify new therapeutic targets designed to prevent proliferation of malignant prostate cells.

Soy isoflavones alter expression of genes associated with cancer progression, including interleukin-8, in androgen-independent PC-3 human prostate cancer cells.

High consumption of soy isoflavones in Asian diets has been correlated with a lower incidence of clinically important cases of prostate cancer. The chemopreventive properties of these diets may result from an interaction of several types of isoflavones, including genistein and daidzein. The present study investigated the effects of a soy isoflavone concentrate (ISF) on growth and gene expression profiles of PC-3 human prostate cancer cells. Trypan blue exclusion and [3H]-thymidine incorporation assays showed that ISF decreased cell viability and caused a dose-dependent inhibition of DNA synthesis, respectively, with 50% inhibition (IC50) of DNA synthesis at 52 mg/L (P = 0.05). The glucoside conjugates of genistein and daidzein in ISF were converted to bioactive free aglycones in cell culture in association with the inhibition of DNA synthesis. Flow cytometry and Western immunoblot analyses showed that ISF at 200 mg/L caused an accumulation of cells in the G2/M phase of the cell cycle (P < 0.05) and decreased cyclin A by 20% (P < 0.05), respectively. The effect of ISF on the gene expression profile of PC-3 cells was analyzed using Affymetrix oligonucleotide DNA microarrays that interrogate approximately 17,000 human genes. Of the 75 genes altered by ISF, 28 were upregulated and 47 were downregulated (P < 0.05). Further analysis showed that IL-8, matrix metalloproteinase 13, inhibin beta A, follistatin, and fibronectin mRNA levels were significantly reduced, whereas the expression of p21(CIP1), a major cell cycle inhibitory protein, was increased. The effects of ISF on the expression of IL-8 and p21(CIP1) mRNA and protein were validated at high and low ISF concentrations. Our data show that ISF inhibits the growth of PC-3 cells through modulation of cell cycle progression and the expression of genes involved in cell cycle regulation, metastasis, and angiogenesis.

Mechanisms of the growth inhibitory effects of the isoflavonoid biochanin A on LNCaP cells and xenografts.

BACKGROUND: Isoflavones inhibit the growth of some types of tumor cells, including prostate adenocarcinoma. This study used LNCaP cells and xenografts to investigate the mechanisms of the antiproliferative effects of biochanin A, a major isoflavone present in red clover but not soy-derived products. METHODS: LNCaP cells were exposed to varying doses of biochanin A to evaluate viability, DNA synthesis, and DNA fragmentation (TUNEL) analysis. Regulation of gene expression was determined by using Western immunoblotting and cDNA microarrays. Anti-tumorigenic effects were evaluated by using athymic mice with LNCaP flank tumors. RESULTS: Biochanin A induced a dose-dependent inhibition of proliferation and [(3)H]thymidine incorporation that correlated with increased DNA fragmentation, indicative of apoptosis. Western blot analyses of cell cycle regulatory proteins revealed that biochanin A significantly decreased expression of cyclin B and p21, whereas flow cytometry showed that cells were accumulating in the G(0)/G(1) phase. cDNA microarray analyses identified 29 down-regulated genes with six reduced below assay detection limits. Eleven genes were up-regulated, including 9 that were undetectable in controls. In mice with LNCaP xenografts, biochanin A significantly reduced tumor size and incidence. CONCLUSION: These results indicate that biochanin A inhibits prostate cancer cell growth through induction of cell cycle arrest and apoptosis. Biochanin A-regulated genes suggest multiple pathways of action. Biochanin A inhibits the incidence and growth of LNCaP xenograft tumors in athymic mice.