Synthesis and anticancer potential of novel xanthone derivatives with 3,6-substituted chains.

In an effort to develop new drug candidates with enhanced anticancer activity, our team synthesized and assessed the cytotoxicity of a series of novel xanthone derivatives with two longer 3,6-disubstituted amine carbonyl methoxy side chains on either benzene ring in selected human cancer cell lines. An MTT assay revealed that a set of compounds with lower IC50 values than the positive control, 5-FU, exhibited greater anticancer effects. The most potent derivative (XD8) exhibited anticancer activity in MDA-MB-231, PC-3, A549, AsPC-1, and HCT116 cells lines with IC50 values of 8.06, 6.18, 4.59, 4.76, and 6.09µM, respectively. Cell cycle analysis and apoptosis activation suggested that the mechanism of action of these derivatives includes cell cycle regulation and apoptosis induction.

A new flavonoid regulates angiogenesis and reactive oxygen species production.

The tumor vascular system, which is critical to the survival and growth of solid tumors, has been an attractive target for anticancer research. Building on studies that show that some flavonoids have anticancer vascular effects, we developed and analyzed the flavonoid derivative R24 [3, 6-bis (2-oxiranylmethoxy)-9H-xanthen-9-one]. A CAM assay revealed that R24 disrupted neovascular formation; fewer dendrites were detected and overall dendritic length was shorter in the R24-treated chicken embryos. The antiproliferative effect of R24 was measured by MTT assay in A549 (lung cancer), AsPC-1 (pancreatic cancer), HCT-116 (colorectal cancer), and PC-3 (prostate cancer) cell lines. R24 reduced proliferation with an IC50 of 3.44, 3.59, 1.22, and 11.83 µM, respectively. Cell-cycle analysis and Annexin-V/propidium iodide staining showed that R24 induced apoptosis. In addition, R24 regulated intracellular ROS production in a dose-dependent manner. CM-H2DCFDA staining indicated that intracellular ROS production increased with the R24 dose. In summary, we found that R24 exhibits potent antiangiogenic and antiproliferative effects, induces apoptosis, and promotes ROS production.

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.

Effects on Lung Tissue After Breast Cancer Radiation: Comparing Photon and Proton Therapies.

Purpose: In breast cancer, improved treatment approaches that reduce injury to lung tissue and early diagnosis and intervention for lung toxicity are increasingly important in survivorship. The aims of this study are to (1) compare lung tissue radiographic changes in women treated with conventional photon radiation therapy and those treated with proton therapy (PT), (2) assess the volume of lung irradiated to 5 Gy (V5) and 20 Gy (V20) by treatment modality, and (3) quantify the effects of V5, V20, time, and smoking history on the severity of tissue radiographic changes. Patients and Methods: A prospective observational study of female breast cancer patients was conducted to monitor postradiation subclinical lung tissue radiographic changes. Repeated follow-up x-ray computed tomography scans were acquired through 2 years after treatment. In-house software was used to quantify an internally normalized measure of pulmonary tissue density change over time from the computed tomography scans, emphasizing the 6- and 12-month time points. Results: Compared with photon therapy, PT was associated with significantly lower lung V5 and V20. Lung V20 (but not V5) correlated significantly with increased subclinical lung tissue radiographic changes 6 months after treatment, and neither correlated with lung effects at 12 months. Significant lung tissue density changes were present in photon therapy patients at 6 and 12 months but not in PT patients. Significant lung tissue density change persisted at 12 months in ever-smokers but not in never-smokers. Conclusion: Patients treated with PT had significantly lower radiation exposure to the lungs and less statistically significant tissue density change, suggesting decreased injury and/or improved recovery compared to photon therapy. These findings motivate additional studies in larger, randomized, and more diverse cohorts to further investigate the contributions of treatment modality and smoking regarding the short- and long-term radiographic effects of radiation on lung tissue.

Maternal bias in mouse radiosensitivity: the role of the mitochondrial PTP.

This study investigated, at the molecular level, mitochondrial responses to radiation. In three mouse strains, we found the following: (1) mitochondrial response to calcium stress was associated with a strain's susceptibility to γ-radiation; (2) γ-radiation increased this calcium stress response in a dose-responsive manner; (3) the mitochondrial DNA (mtDNA) copy number in the liver of the radiosensitive mouse strain was significantly lower, as compared to that of the radioresistant strain; (4) adenine nucleotide translocase (ANT) mRNA copy numbers were significantly lower in the radiosensitive strain; (5) the F1 offspring (BC/C57M) of radiosensitive females mated with radioresistant males exhibited a significant difference in calcium stress response from that of the radiation-resistant strain, but the reverse cross did not exhibit this difference; and (6) only those mitochondria extracted from the livers of irradiated BC/C57M mice exhibited a heightened calcium stress response. We propose that a genetic change in ANT and a postirradiation change involving either mtDNA-encoded protein replacement or altered mtDNA association fit these data.

Implications of "flash" radiotherapy for biodosimetry.

Extremely high dose rate radiation delivery (FLASH) for cancer treatment has been shown to produce less damage to normal tissues while having the same radiotoxic effect on tumor tissue (referred to as the FLASH effect). Research on the FLASH effect has two very pertinent implications for the field of biodosimetry: (1) FLASH is a good model to simulate delivery of prompt radiation from the initial moments after detonating a nuclear weapon and (2) the FLASH effect elucidates how dose rate impacts the biological mechanisms that underlie most types of biological biodosimetry. The impact of dose rate will likely differ for different types of biodosimetry, depending on the specific underlying mechanisms. The greatest impact of FLASH effects is likely to occur for assays based on biological responses to radiation damage, but the consequences of differential effects of dose rates on the accuracy of dose estimates has not been taken into account.

Benefits and challenges of in vivo EPR nail biodosimetry in a second tier of medical triage in response to a large radiation event.

Following large-scale radiation events, an overwhelming number of people will potentially need mitigators or treatment for radiation-induced injuries. This necessitates having methods to triage people based on their dose and its likely distribution, so life-saving treatment is directed only to people who can benefit from such care. Using estimates of victims following an improvised nuclear device striking a major city, we illustrate a two-tier approach to triage. At the second tier, after first removing most who would not benefit from care, biodosimetry should provide accurate dose estimates and determine whether the dose was heterogeneous. We illustrate the value of using in vivo electron paramagnetic resonance nail biodosimetry to rapidly assess dose and determine its heterogeneity using independent measurements of nails from the hands and feet. Having previously established its feasibility, we review the benefits and challenges of potential improvements of this method that would make it particularly suitable for tier 2 triage. Improvements, guided by a user-centered approach to design and development, include expanding its capability to make simultaneous, independent measurements and improving its precision and universality.

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.

Measuring Radiation Toxicity Using Circulating Cell-Free DNA in Prostate Cancer Patients.

BACKGROUND: After radiation therapy (RT), circulating plasma cell-free DNA (cfDNA) released in response to RT damage to tissue can be measured within hours. We examined for a correlation between cfDNA measured during the first week of therapy and early and late gastrointestinal (GI) and genitourinary (GU) toxicity. MATERIAL AND METHODS: Patients were eligible for enrollment if they planned to receive proton or photon RT for nonmetastatic prostate cancer in the setting of an intact prostate or after prostatectomy. Blood was collected before treatment and on sequential treatment days for the first full week of therapy. Toxicity assessments were performed at baseline, weekly during RT, and 6 months and 12 months after RT. Data were analyzed to examine correlations among patient-reported GI and GU toxicities. RESULTS: Fifty-four patients were evaluable for this study. Four (7%) and 3 (6%) patients experienced acute and late grade 2 GI toxicity, respectively. Twenty-two (41%) and 18 (35%) patients experienced acute and late grade 2 GU toxicity, respectively. No patients developed grade 3 or higher toxicity. Grade 2 acute GI toxicity, but not grade 2 acute GU toxicity, was significantly correlated with pre-RT cfDNA levels and on all days 1, 2, 3, 4, and 5 of RT (P < .005). Grade 2 late GI toxicity, but not GU toxicity, was significantly correlated with pre-RT cfDNA levels (P = .021). CONCLUSIONS: Based on this preliminary study, cfDNA levels can potentially predict the subset of patients destined to develop GI toxicity during prostate cancer treatment. Given that the toxicity profiles of the various fractionations and modalities are highly similar, the data support the expectation that cfDNA could provide a biological estimate to complement the dose-volume histogram. A test of this hypothesis is under evaluation in a National Cancer Institute-funded multi-institutional study.

Antioxidant properties of select radiation mitigators based on semicarbazone and pyrazole derivatives of curcumin.

Fifty-eight semicarbazone and pyrazole derivatives of curcumin have been developed as potential mitigation agents to treat acute radiation syndrome (ARS). Pyridyl (D12, D13), furyl (D56), and phenyl (D68) derivatives of curcumin semi-carbazones were found to provide the highest dose modifying factors (DMF) with respect to survival in sub-TBI (bone marrow sparing) exposures in mouse models. To investigate the basis for the mitigating effects of these agents on ARS, we examined their oxidation potentials and radical scavenging properties in comparison to other semicarbazone and pyrazole curcumin derivatives with less effective DMFs. Comparisons between D12, D13, D56, and D68 and other semicarbazone and pyrazole derivatives of curcumin did not show a sufficient difference in reducing properties and hydrogen atom donating properties for these properties to be the basis of the dose modifying activities of these compounds. Therefore, their DMFs likely reflect structure-activity relationship(s),wherein interaction with key receptors or alteration of enzyme expression result in modifications of cellular or tissue responses to radiation, rather than on the derivatives' ability to modify radiation-induced flux of free radicals through direct interaction with these radicals.

Antioxidant properties of quercetin.

UNLABELLED: Quercetin, a plant-derived aglycone form of flavonoid glycosides, has been used as a nutritional supplement and may be beneficial against a variety of diseases, including cancer. We examined the antioxidant properties of quercetin. The reduction potential of quercetin was measured at various pH values using voltammetric methods, and its total antioxidant capacity (TAC) was measured using the phosphomolybdenum method. The effect of quercetin on production of reactive oxygen species (ROS) and nitric oxide (NO) in LPS-stimulated human THP-1 acute monocytic leukemia cells was determined by flow cytometry using CM-H2DCFDA dye. The results were compared with curcumin, a natural product exhibiting a similar range of reported health benefits. RESULTS: 1) Quercetin has a higher reduction potential compared with curcumin at three different pH settings and is comparable to Trolox at pH 7-9.5; 2) its TAC is 3.5 fold higher than curcumin; 3) it reduced LPS-induced ROS to near normal levels; 4) it reduced LPS-induced NO production. These data provide a physico-chemical basis for comparing antioxidants, with potential benefits individually or in combination.

Advances towards using finger/toenail dosimetry to triage a large population after potential exposure to ionizing radiation.

Rapid and accurate retrospective dosimetry is of critical importance and strategic value for the emergency medical response to a large-scale radiological/nuclear event. One technique that has the potential for rapid and accurate dosimetry measurements is electron paramagnetic resonance (EPR) spectroscopy of relatively stable radiation-induced signals (RIS) in fingernails and toenails. Two approaches are being developed for EPR nail dosimetry. In the approach using ex vivo measurements on nail clippings, accurate estimation of the dose-dependent amplitude of the RIS is complicated by the presence of mechanically-induced signals (MIS) that are generated during the nail clipping. Recent developments in ex vivo nail dosimetry, including a thorough characterization of the MIS and an appreciation of the role of hydration and the development of effective analytic techniques, have led to improvements in the accuracy and precision of this approach. An in vivo nail dosimetry approach is also very promising, as it eliminates the problems of MIS from the clipping and it has the potential to be an effective and efficient approach for field deployment. Two types of EPR resonators are being developed for in vivo measurements of fingernails and toenails.

An analysis of the effects of Mn2+ on oxidative phosphorylation in liver, brain, and heart mitochondria using state 3 oxidation rate assays.

Manganese (Mn) toxicity is partially mediated by reduced ATP production. We have used oxidation rate assays--a measure of ATP production--under rapid phosphorylation conditions to explore sites of Mn(2+) inhibition of ATP production in isolated liver, brain, and heart mitochondria. This approach has several advantages. First, the target tissue for Mn toxicity in the basal ganglia is energetically active and should be studied under rapid phosphorylation conditions. Second, Mn may inhibit metabolic steps which do not affect ATP production rate. This approach allows identification of inhibitions that decrease this rate. Third, mitochondria from different tissues contain different amounts of the components of the metabolic pathways potentially resulting in different patterns of ATP inhibition. Our results indicate that Mn(2+) inhibits ATP production with very different patterns in liver, brain, and heart mitochondria. The primary Mn(2+) inhibition site in liver and heart mitochondria, but not in brain mitochondria, is the F1F0 ATP synthase. In mitochondria fueled by either succinate or glutamate+malate, ATP production is much more strongly inhibited in brain than in liver or heart mitochondria; moreover, Mn(2+) inhibits two independent sites in brain mitochondria. The primary site of Mn-induced inhibition of ATP production in brain mitochondria when succinate is substrate is either fumarase or complex II, while the likely site of the primary inhibition when glutamate plus malate are the substrates is either the glutamate/aspartate exchanger or aspartate aminotransferase.

Scientific and Logistical Considerations When Screening for Radiation Risks by Using Biodosimetry Based on Biological Effects of Radiation Rather than Dose: The Need for Prior Measurements of Homogeneity and Distribution of Dose.

An effective medical response to a large-scale radiation event requires prompt and effective initial triage so that appropriate care can be provided to individuals with significant risk for severe acute radiation injury. Arguably, it would be advantageous to use injury rather than radiation dose for the initial assessment; i.e., use bioassays of biological damage. Such assays would be based on changes in intrinsic biological response elements; e.g., up- or down-regulation of genes, proteins, metabolites, blood cell counts, chromosomal aberrations, micronuclei, micro-RNA, cytokines, or transcriptomes. Using a framework to evaluate the feasibility of biodosimetry for triaging up to a million people in less than a week following a major radiation event, Part 1 analyzes the logistical feasibility and clinical needs for ensuring that biomarkers of organ-specific injury could be effectively used in this context. We conclude that the decision to use biomarkers of organ-specific injury would greatly benefit by first having independent knowledge of whether the person's exposure was heterogeneous and, if so, what was the dose distribution (to determine which organs were exposed to high doses). In Part 2, we describe how these two essential needs for prior information (heterogeneity and dose distribution) could be obtained by using in vivo nail dosimetry. This novel physical biodosimetry method can also meet the needs for initial triage, providing non-invasive, point-of-care measurements made by non-experts with immediate dose estimates for four separate anatomical sites. Additionally, it uniquely provides immediate information as to whether the exposure was homogeneous and, if not, it can estimate the dose distribution. We conclude that combining the capability of methods such as in vivo EPR nail dosimetry with bioassays to predict organ-specific damage would allow effective use of medical resources to save lives.

Circulating Cell-Free DNA Correlates with Body Integral Dose and Radiation Modality in Prostate Cancer.

PURPOSE: The RadTox assay measures circulating cell-free DNA released in response to radiotherapy (RT)-induced tissue damage. The primary objectives for this clinical trial were to determine whether cell-free DNA numbers measured by the RadTox assay are (1) correlated with body integral dose, (2) lower with proton RT compared with photon RT, and (3) higher with larger prostate cancer RT fields. PATIENTS AND METHODS: Patients planned to receive proton or photon RT for nonmetastatic prostate cancer in the setting of an intact prostate or postprostatectomy were eligible for the trial. Plasma was collected pre-RT and at 5 additional daily collection points beginning 24 hours after the initiation of RT. Data from 54 evaluable patients were analyzed to examine any correlations among RadTox scores with body-integral dose, RT modality (photon versus proton), and RT field size (prostate or prostate bed versus whole pelvis). RESULTS: Body integral dose was significantly associated with the peak post-RT RadTox score (P = .04). Patients who received photon RT had a significant increase in peak post-RT RadTox score (P = .04), average post-RT RadTox score (P = .04), and day-2 RadTox score (all minus the pre-RT values for each patient) as compared with patients who received proton RT. Field size was not significantly associated with RadTox score. CONCLUSION: RadTox is correlated with body integral dose and correctly predicts which patients receive proton versus photon RT. Data collection remains ongoing for patient-reported RT toxicity outcomes to determine whether RadTox scores are correlated with toxicity.

Replication of murine mitochondrial DNA following irradiation.

The effect of radiation on the mitochondrial genome in vivo is largely unknown. Though mitochondrial DNA (mtDNA) is vital for cellular survival and proliferation, it has little DNA repair machinery compared with nuclear DNA (nDNA). A better understanding of how radiation affects mtDNA should lead to new approaches for radiation protection. We have developed a new system using real-time PCR that sensitively detects the change in copy number of mtDNA compared with nDNA. In each sample, the DNA sequence coding 18S rRNA served as the nDNA reference in a run simultaneously with a mtDNA sequence. Small bowel collected 24 hours after 2 Gy or 4 Gy total body irradiation (TBI) exhibited increased levels of mtDNA compared with control mice. A 4 Gy dose produced a greater effect than 2 Gy. Similarly, in bone marrow collected 24 hours after 4 Gy or 7 Gy TBI, 7 Gy produced a greater response than 4 Gy. As a function of time, a greater effect was seen at 48 hours compared with 24 hours. In conclusion, we found that radiation increased the ratio of mtDNA:nDNA and that this effect seems to be tissue independent and seems to increase with radiation dose and duration following radiation exposure.

Bioanalytical method development and validation for determination of fibroblast growth factor peptide and its application to pharmacokinetic studies.

Fibroblast growth factor peptide (FGF-P) is a polypeptide analog of FGF-2 that could be a potential mitigation and treatment agent for radiation syndromes. Prior to conducting preclinical pharmacokinetics, we developed and validated the LC-MS/MS bioanalytical method for determination of FGF-P in rat plasma for the first time. FGF-P was extracted from rat plasma using the protein precipitation technique followed liquid-liquid extraction using dichloromethane as a solvent. The mobile phases consisted of two components: (a) 0.1% formic acid in water; and (b) acetonitrile: 0.1% formic acid in water (95:5) under gradient elution. The validated method was also successfully applied to a pharmacokinetic study of FGF-P (10 mg/kg, intravenous) in Wistar rats. The method proved to be specific, accurate, precise, and linear over the concentration range of 2-500 ng/mL with coefficient of determination greater than 0.99 in all validation batches. The within-run and between-run accuracy was 87.97-115.00% with a precision of less than 14%. The mean recoveries ranged from 88.14% to 101.73%. The stability of the compound in plasma samples was proven under various storage conditions. After intravenous administration of FGF-P (10 mg/kg) the C0 was 70.4 µg/mL and the AUC was 86.2 µg*min/mL.

Radiation-induced tumor immunity in patients with non-small cell lung cancer.

BACKGROUND: Radiation-induced tumor immunity (RITI) influences primary tumor growth and development of metastases in preclinical cancer models with conventional radiotherapy. Antigen-specific immune responses have also been shown for prostate cancer treated with radiotherapy. We examined whether RITI can be induced in patients with non-small cell lung cancer (NSCLC) following proton radiotherapy. METHODS: Pre- and post-radiotherapy plasma samples from 26 patients with nonmetastatic NSCLC who received radiotherapy between 2010 and 2012 were evaluated by western blotting for IgG and IgM bands to assess RITI response to tumor antigens from lung cancer cell lines. Statistical analysis was used to evaluate any correlation among IgG or IgM and clinical outcomes. RESULTS: Twenty-one patients received proton therapy at 2 GyRBE/fraction (n = 17) or 6-12 Gy/fraction (n = 4); five received photon therapy at 2-2.5 GyRBE/fraction. Compared with the pretreatment baseline, new IgG or IgM binding was detected in 27% and 50% of patients, respectively. New IgG bands were detected in the 25-37 kD, 50-75 kD, and 75-100 kD ranges. New IgM bands were detected in the 20-25 kD, 25-37 kD, 37-50 kD, 50-75 kD, and 75-100 kD ranges. There was no difference in IgG and/or IgM RITI response in patients treated with photons versus protons, or in patients who received SBRT compared to standard fractionation (P > 0.05). There was no difference in overall survival, metastasis-free survival, or local control based on IgG and/or IgM RITI response (P > 0.05). CONCLUSION: RITI can be induced in patients with NSCLC through upregulated IgG and/or IgM. RITI response was not associated with proton versus photon therapy or with clinical outcomes in this small cohort and should be examined in a larger cohort in future studies.