Natural 2',4-Dihydroxy-4',6'-dimethoxy Chalcone Isolated from Chromolaena tacotana Inhibits Breast Cancer Cell Growth through Autophagy and Mitochondrial Apoptosis.

Breast cancer (BC) is one of the most common cancers among women. Effective treatment requires precise tailoring to the genetic makeup of the cancer for improved efficacy. Numerous research studies have concentrated on natural compounds and their anti-breast cancer properties to improve the existing treatment options. Chromolaena tacotana (Klatt) R.M. King and H. Rob (Ch. tacotana) is a notable source of bioactive hydroxy-methylated flavonoids. However, the specific anti-BC mechanisms of these flavonoids, particularly those present in the plant's inflorescences, remain partly undefined. This study focuses on assessing a chalcone derivative extracted from Ch. tacotana inflorescences for its potential to concurrently activate regulated autophagy and intrinsic apoptosis in luminal A and triple-negative BC cells. We determined the chemical composition of the chalcone using ultraviolet (UV) and nuclear magnetic resonance (NMR) spectroscopy. Its selective cytotoxicity against BC cell lines was assessed using the MTT assay. Flow cytometry and Western blot analysis were employed to examine the modulation of proteins governing autophagy and the intrinsic apoptosis pathway. Additionally, in silico simulations were conducted to predict interactions between chalcone and various anti-apoptotic proteins, including the mTOR protein. Chalcone was identified as 2',4-dihydroxy-4',6'-dimethoxy-chalcone (DDC). This compound demonstrated a selective inhibition of BC cell proliferation and triggered autophagy and intrinsic apoptosis. It induced cell cycle arrest in the G0/G1 phase and altered mitochondrial outer membrane potential (∆ψm). The study detected the activation of autophagic LC3-II and mitochondrial pro-apoptotic proteins in both BC cell lines. The regulation of Bcl-XL and Bcl-2 proteins varied according to the BC subtype, yet they showed promising molecular interactions with DDC. Among the examined pro-survival proteins, mTOR and Mcl-1 exhibited the most favorable binding energies and were downregulated in BC cell lines. Further research is needed to fully understand the molecular dynamics involved in the activation and interaction of autophagy and apoptosis pathways in cancer cells in response to potential anticancer agents, like the hydroxy-methylated flavonoids from Ch. tacotana.

A Novel Tri-Hydroxy-Methylated Chalcone Isolated from Chromolaena tacotana with Anti-Cancer Potential Targeting Pro-Survival Proteins.

Chromolaena tacotana (Klatt) R. M. King and H. Rob (Ch. tacotana) contains bioactive flavonoids that may have antioxidant and/or anti-cancer properties. This study investigated the potential anti-cancer properties of a newly identified chalcone isolated from the inflorescences of the plant Chromolaena tacotana (Klatt) R. M. King and H. Rob (Ch. tacotana). The chalcone structure was determined using HPLC/MS (QTOF), UV, and NMR spectroscopy. The compound cytotoxicity and selectivity were evaluated on prostate, cervical, and breast cancer cell lines using the MTT assay. Apoptosis and autophagy induction were assessed through flow cytometry by detecting annexin V/7-AAD, active Casp3/7, and LC3B proteins. These results were supported by Western blot analysis. Mitochondrial effects on membrane potential, as well as levels of pro- and anti-apoptotic proteins were analyzed using flow cytometry, fluorescent microscopy, and Western blot analysis specifically on a triple-negative breast cancer (TNBC) cell line. Furthermore, molecular docking (MD) and molecular dynamics (MD) simulations were performed to evaluate the interaction between the compounds and pro-survival proteins. The compound identified as 2',3,4-trihydroxy-4',6'-dimethoxy chalcone inhibited the cancer cell line proliferation and induced apoptosis and autophagy. MDA-MB-231, a TNBC cell line, exhibited the highest sensitivity to the compound with good selectivity. This activity was associated with the regulation of mitochondrial membrane potential, activation of the pro-apoptotic proteins, and reduction of anti-apoptotic proteins, thereby triggering the intrinsic apoptotic pathway. The chalcone consistently interacted with anti-apoptotic proteins, particularly the Bcl-2 protein, throughout the simulation period. However, there was a noticeable conformational shift observed with the negative autophagy regulator mTOR protein. Future studies should focus on the molecular mechanisms underlying the anti-cancer potential of the new chalcone and other flavonoids from Ch. tacotana, particularly against predominant cancer cell types.

An Immunoinformatics Approach for SARS-CoV-2 in Latam Populations and Multi-Epitope Vaccine Candidate Directed towards the World's Population.

The coronavirus pandemic is a major public health crisis affecting global health systems with dire socioeconomic consequences, especially in vulnerable regions such as Latin America (LATAM). There is an urgent need for a vaccine to help control contagion, reduce mortality and alleviate social costs. In this study, we propose a rational multi-epitope candidate vaccine against SARS-CoV-2. Using bioinformatics, we constructed a library of potential vaccine peptides, based on the affinity of the most common major human histocompatibility complex (HLA) I and II molecules in the LATAM population to predict immunological complexes among antigenic, non-toxic and non-allergenic peptides extracted from the conserved regions of 92 proteomes. Although HLA-C, had the greatest antigenic peptide capacity from SARS-CoV-2, HLA-B and HLA-A, could be more relevant based on COVID-19 risk of infection in LATAM countries. We also used three-dimensional structures of SARS-CoV-2 proteins to identify potential regions for antibody production. The best HLA-I and II predictions (with increased coverage in common alleles and regions evoking B lymphocyte responses) were grouped into an optimized final multi-epitope construct containing the adjuvants Beta defensin-3, TpD, and PADRE, which are recognized for invoking a safe and specific immune response. Finally, we used Molecular Dynamics to identify the multi-epitope construct which may be a stable target for TLR-4/MD-2. This would prove to be safe and provide the physicochemical requirements for conducting experimental tests around the world.

In vivo bioluminescence tomography-guided radiation research platform for pancreatic cancer: an initial study using subcutaneous and orthotopic pancreatic tumor models.

Genetically engineered mouse model(GEMM) that develops pancreatic ductal adenocarcinoma(PDAC) offers an experimental system to advance our understanding of radiotherapy(RT) for pancreatic cancer. Cone beam CT(CBCT)-guided small animal radiation research platform(SARRP) has been developed to mimic the RT used for human. However, we recognized that CBCT is inadequate to localize the PDAC growing in low image contrast environment. We innovated bioluminescence tomography(BLT) to guide SARRP irradiation for in vivo PDAC. Before working on the complex PDAC-GEMM, we first validated our BLT target localization using subcutaneous and orthotopic pancreatic tumor models. Our BLT process involves the animal transport between the BLT system and SARRP. We inserted a titanium wire into the orthotopic tumor as the fiducial marker to track the tumor location and to validate the BLT reconstruction accuracy. Our data shows that with careful animal handling, minimum disturbance for target position was introduced during our BLT imaging procedure(<0.5mm). However, from longitudinal 2D bioluminescence image(BLI) study, the day-to-day location variation for an abdominal tumor can be significant. We also showed that the 2D BLI in single projection setting cannot accurately capture the abdominal tumor location. It renders that 3D BLT with multiple-projection is needed to quantify the tumor volume and location for precise radiation research. Our initial results show the BLT can retrieve the location at 2mm accuracy for both tumor models, and the tumor volume can be delineated within 25% accuracy. The study for the subcutaneous and orthotopic models will provide us valuable knowledge for BLT-guided PDAC-GEMM radiation research.

Uncovering the Role of N-Acetyl-Aspartyl-Glutamate as a Glutamate Reservoir in Cancer.

N-acetyl-aspartyl-glutamate (NAAG) is a peptide-based neurotransmitter that has been extensively studied in many neurological diseases. In this study, we show a specific role of NAAG in cancer. We found that NAAG is more abundant in higher grade cancers and is a source of glutamate in cancers expressing glutamate carboxypeptidase II (GCPII), the enzyme that hydrolyzes NAAG to glutamate and N-acetyl-aspartate (NAA). Knocking down GCPII expression through genetic alteration or pharmacological inhibition of GCPII results in a reduction of both glutamate concentrations and cancer growth. Moreover, targeting GCPII in combination with glutaminase inhibition accentuates these effects. These findings suggest that NAAG serves as an important reservoir to provide glutamate to cancer cells through GCPII when glutamate production from other sources is limited. Thus, GCPII is a viable target for cancer therapy, either alone or in combination with glutaminase inhibition.

Dynamic Effects of CYP2D6 Genetic Variants in a Set of Poor Metaboliser Patients with Infiltrating Ductal Cancer Under Treatment with Tamoxifen.

Breast cancer is a group of multigenic diseases. It is the most common cancer diagnosed among women worldwide and is often treated with tamoxifen. Tamoxifen is catalysed by cytochrome P450 2D6 (CYP2D6), and inter-individual variations in the enzyme due to single nucleotide polymorphisms (SNPs) could alter enzyme activity. We evaluated SNPs in patients from Colombia in South America who were receiving tamoxifen treatment for breast cancer. Allelic diversity in the CYP2D6 gene was found in the studied population, with two patients displaying the poor-metaboliser phenotype. Molecular dynamics and trajectory analyses were performed for CYP2D6 from these two patients, comparing it with the common allelic form (CYP2D6*1). Although we found no significant structural change in the protein, its dynamics differ significantly from those of CYP2D6*1, the effect of such differential dynamics resulting in an inefficient enzyme with serious implications for tamoxifen-treated patients, increasing the risk of disease relapse and ineffective treatment.

Preclinical Benefit of Hypoxia-Activated Intra-arterial Therapy with Evofosfamide in Liver Cancer.

PURPOSE: To evaluate safety and characterize anticancer efficacy of hepatic hypoxia-activated intra-arterial therapy (HAIAT) with evofosfamide in a rabbit model. EXPERIMENTAL DESIGN: VX2-tumor-bearing rabbits were assigned to 4 intra-arterial therapy (IAT) groups (n = 7/group): (i) saline (control); (ii) evofosfamide (Evo); (iii) doxorubicin-lipiodol emulsion followed by embolization with 100-300 µm beads (conventional, cTACE); or (iv) cTACE and evofosfamide (cTACE + Evo). Blood samples were collected pre-IAT and 1, 2, 7, and 14 days post-IAT. A semiquantitative scoring system assessed hepatocellular damage. Tumor volumes were segmented on multidetector CT (baseline, 7/14 days post-IAT). Pathologic tumor necrosis was quantified using manual segmentation on whole-slide images. Hypoxic fraction (HF) and compartment (HC) were determined by pimonidazole staining. Tumor DNA damage, apoptosis, cell proliferation, endogenous hypoxia, and metabolism were quantified (γ-H2AX, Annexin V, caspase-3, Ki-67, HIF1α, VEGF, GAPDH, MCT4, and LDH). RESULTS: cTACE + Evo showed a similar profile of liver enzymes elevation and pathologic scores compared with cTACE. Neither hematologic nor renal toxicity were observed. Animals treated with cTACE + Evo demonstrated smaller tumor volumes, lower tumor growth rates, and higher necrotic fractions compared with cTACE. cTACE + Evo resulted in a marked reduction in the HF and HC. Correlation was observed between decreases in HF or HC and tumor necrosis. cTACE + Evo promoted antitumor effects as evidenced by increased expression of γ-H2AX, apoptotic biomarkers, and decreased cell proliferation. Increased HIF1α/VEGF expression and tumor glycolysis supported HAIAT. CONCLUSIONS: HAIAT achieved a promising step towards the locoregional targeting of tumor hypoxia. The favorable toxicity profile and enhanced anticancer effects of evofosfamide in combination with cTACE pave the way towards clinical trials in patients with liver cancer. Clin Cancer Res; 23(2); 536-48. ©2016 AACR.

Evaluation of On- and Off-Line Bioluminescence Tomography System for Focal Irradiation Guidance.

In response to the limitations of computed tomography (CT) and cone-beam CT (CBCT) in irradiation guidance, especially for soft-tissue targets without the use of contrast agents, our group developed a solution that implemented bioluminescence tomography (BLT) as the image-guidance modality for preclinical radiation research. However, adding such a system to existing small animal irradiators is no small task. A potential solution is to utilize an off-line BLT system in close proximity to the irradiator, with stable and effective animal transport between the two systems. In this study, we investigated the localization accuracy of an off-line BLT system when used for the small animal radiation research platform (SARRP) and compared the results with those of an on-line system. The CBCT was equipped on both the off-line BLT system and the SARRP, with a distance of 5 m between them. To evaluate the setup error during animal transport between the two systems, the mice underwent CBCT imaging on the SARRP and were then transported to the off-line system for a second CBCT imaging session. The normalized intensity difference of the two images and the corresponding histogram and correlation were computed to evaluate if the transport process perturbed animal positioning. Strong correlation (correlation coefficients >0.95) between the SARRP and the off-line mouse CBCT was observed. The offset of the implanted light source center can be maintained within 0.2 mm during transport. To compare the target localization accuracy using the on-line SARRP BLT and the off-line system, a self-illuminated bioluminescent source was implanted in the abdomen of anesthetized mice. In addition to the application for dose calculation, CBCT imaging was also employed to generate the mesh grid of the imaged mouse for BLT reconstruction. Two scenarios were devised and compared, which involved localization of the luminescence source based on either: 1. on-line SARRP bioluminescence image and CBCT; or 2. off-line bioluminescence image and SARRP CBCT. The first scenario is assumed to have the least setup error, because no animal transport was involved. The second scenario examines if an off-line BLT system, with the mesh generated from the SARRP CBCT, can be used to guide SARRP irradiation when there is minimal target contrast in CBCT. Stability during animal transport between the two systems was maintained. The center of mass (CoM) of the light source reconstructed by the off-line BLT had an offset of 1.0 ± 0.4 mm from the true CoM derived from the SARRP CBCT. These results are comparable to the offset of 1.0 ± 0.2 mm using on-line BLT. With CBCT information provided by the SARRP and effective animal immobilization during transport, these findings support the utilization of an off-line BLT-guided system, in close proximity to the SARRP, for accurate soft-tissue target localization. In addition, a dedicated standalone BLT system for our partner site at the University of Pennsylvania was introduced in this study.

Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer.

Targeting glutamine metabolism via pharmacological inhibition of glutaminase has been translated into clinical trials as a novel cancer therapy, but available drugs lack optimal safety and efficacy. In this study, we used a proprietary emulsification process to encapsulate bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a selective but relatively insoluble glutaminase inhibitor, in nanoparticles. BPTES nanoparticles demonstrated improved pharmacokinetics and efficacy compared with unencapsulated BPTES. In addition, BPTES nanoparticles had no effect on the plasma levels of liver enzymes in contrast to CB-839, a glutaminase inhibitor that is currently in clinical trials. In a mouse model using orthotopic transplantation of patient-derived pancreatic tumor tissue, BPTES nanoparticle monotherapy led to modest antitumor effects. Using the HypoxCR reporter in vivo, we found that glutaminase inhibition reduced tumor growth by specifically targeting proliferating cancer cells but did not affect hypoxic, noncycling cells. Metabolomics analyses revealed that surviving tumor cells following glutaminase inhibition were reliant on glycolysis and glycogen synthesis. Based on these findings, metformin was selected for combination therapy with BPTES nanoparticles, which resulted in significantly greater pancreatic tumor reduction than either treatment alone. Thus, targeting of multiple metabolic pathways, including effective inhibition of glutaminase by nanoparticle drug delivery, holds promise as a novel therapy for pancreatic cancer.

Systematic study of target localization for bioluminescence tomography guided radiation therapy.

PURPOSE: To overcome the limitation of CT/cone-beam CT (CBCT) in guiding radiation for soft tissue targets, the authors developed a spectrally resolved bioluminescence tomography (BLT) system for the small animal radiation research platform. The authors systematically assessed the performance of the BLT system in terms of target localization and the ability to resolve two neighboring sources in simulations, tissue-mimicking phantom, and in vivo environments. METHODS: Multispectral measurements acquired in a single projection were used for the BLT reconstruction. The incomplete variables truncated conjugate gradient algorithm with an iterative permissible region shrinking strategy was employed as the optimization scheme to reconstruct source distributions. Simulation studies were conducted for single spherical sources with sizes from 0.5 to 3 mm radius at depth of 3-12 mm. The same configuration was also applied for the double source simulation with source separations varying from 3 to 9 mm. Experiments were performed in a standalone BLT/CBCT system. Two self-illuminated sources with 3 and 4.7 mm separations placed inside a tissue-mimicking phantom were chosen as the test cases. Live mice implanted with single-source at 6 and 9 mm depth, two sources at 3 and 5 mm separation at depth of 5 mm, or three sources in the abdomen were also used to illustrate the localization capability of the BLT system for multiple targets in vivo. RESULTS: For simulation study, approximate 1 mm accuracy can be achieved at localizing center of mass (CoM) for single-source and grouped CoM for double source cases. For the case of 1.5 mm radius source, a common tumor size used in preclinical study, their simulation shows that for all the source separations considered, except for the 3 mm separation at 9 and 12 mm depth, the two neighboring sources can be resolved at depths from 3 to 12 mm. Phantom experiments illustrated that 2D bioluminescence imaging failed to distinguish two sources, but BLT can provide 3D source localization with approximately 1 mm accuracy. The in vivo results are encouraging that 1 and 1.7 mm accuracy can be attained for the single-source case at 6 and 9 mm depth, respectively. For the 2 sources in vivo study, both sources can be distinguished at 3 and 5 mm separations, and approximately 1 mm localization accuracy can also be achieved. CONCLUSIONS: This study demonstrated that their multispectral BLT/CBCT system could be potentially applied to localize and resolve multiple sources at wide range of source sizes, depths, and separations. The average accuracy of localizing CoM for single-source and grouped CoM for double sources is approximately 1 mm except deep-seated target. The information provided in this study can be instructive to devise treatment margins for BLT-guided irradiation. These results also suggest that the 3D BLT system could guide radiation for the situation with multiple targets, such as metastatic tumor models.

Bioluminescence Tomography-Guided Radiation Therapy for Preclinical Research.

PURPOSE: In preclinical radiation research, it is challenging to localize soft tissue targets based on cone beam computed tomography (CBCT) guidance. As a more effective method to localize soft tissue targets, we developed an online bioluminescence tomography (BLT) system for small-animal radiation research platform (SARRP). We demonstrated BLT-guided radiation therapy and validated targeting accuracy based on a newly developed reconstruction algorithm. METHODS AND MATERIALS: The BLT system was designed to dock with the SARRP for image acquisition and to be detached before radiation delivery. A 3-mirror system was devised to reflect the bioluminescence emitted from the subject to a stationary charge-coupled device (CCD) camera. Multispectral BLT and the incomplete variables truncated conjugate gradient method with a permissible region shrinking strategy were used as the optimization scheme to reconstruct bioluminescent source distributions. To validate BLT targeting accuracy, a small cylindrical light source with high CBCT contrast was placed in a phantom and also in the abdomen of a mouse carcass. The center of mass (CoM) of the source was recovered from BLT and used to guide radiation delivery. The accuracy of the BLT-guided targeting was validated with films and compared with the CBCT-guided delivery. In vivo experiments were conducted to demonstrate BLT localization capability for various source geometries. RESULTS: Online BLT was able to recover the CoM of the embedded light source with an average accuracy of 1 mm compared to that with CBCT localization. Differences between BLT- and CBCT-guided irradiation shown on the films were consistent with the source localization revealed in the BLT and CBCT images. In vivo results demonstrated that our BLT system could potentially be applied for multiple targets and tumors. CONCLUSIONS: The online BLT/CBCT/SARRP system provides an effective solution for soft tissue targeting, particularly for small, nonpalpable, or orthotopic tumor models.

HSO3Cl: a prototype molecule for studying OH-stretching overtone induced photodissociation.

Vibrationally induced photodissociation in sulfurochloridic acid (HSO3Cl) is found to be a viable process to form SO3 and HCl from excitations of the OH-stretching overtone starting at νOH = 4. Reactive molecular dynamics simulations on a fully-dimensional potential energy surface fitted to MP2 calculations show that hydrogen transfer and HCl elimination compete with one another on the nanosecond time scale. Excitation with 5 and 6 quanta in the OH-stretch direct elimination of HCl is a dominant process on the several hundred picosecond time scale. At longer times, HCl formation is preceded by intramolecular hydrogen transfer and concomitant excitation of torsional degrees of freedom. As HSO3Cl is a suitable proxy for H2SO4, which is relevant for weather and climate in the upper atmosphere, it is concluded that vibrationally induced photodissociation is a possible mechanism for H2SO4 decomposition. Final state energy distributions for different internal degrees of freedom are predicted which should be observable in laboratory measurements.

Systemic delivery of microencapsulated 3-bromopyruvate for the therapy of pancreatic cancer.

PURPOSE: This study characterized the therapeutic efficacy of a systemically administered formulation of 3-bromopyruvate (3-BrPA), microencapsulated in a complex with ß-cyclodextrin (ß-CD), using an orthotopic xenograft mouse model of pancreatic ductal adenocarcinoma (PDAC). EXPERIMENTAL DESIGN: The presence of the ß-CD-3-BrPA complex was confirmed using nuclear magnetic resonance spectroscopy. Monolayer as well as three-dimensional organotypic cell culture was used to determine the half-maximal inhibitory concentrations (IC50) of ß-CD-3-BrPA, free 3-BrPA, ß-CD (control), and gemcitabine in MiaPaCa-2 and Suit-2 cell lines, both in normoxia and hypoxia. Phase-contrast microscopy, bioluminescence imaging (BLI), as well as zymography and Matrigel assays were used to characterize the effects of the drug in vitro. An orthotopic lucMiaPaCa-2 xenograft tumor model was used to investigate the in vivo efficacy. RESULTS: ß-CD-3-BrPA and free 3-BrPA demonstrated an almost identical IC50 profile in both PDAC cell lines with higher sensitivity in hypoxia. Using the Matrigel invasion assay as well as zymography, 3-BrPA showed anti-invasive effects in sublethal drug concentrations. In vivo, animals treated with ß-CD-3-BrPA demonstrated minimal or no tumor progression as evident by the BLI signal as opposed to animals treated with gemcitabine or the ß-CD (60-fold and 140-fold signal increase, respectively). In contrast to animals treated with free 3-BrPA, no lethal toxicity was observed for ß-CD-3-BrPA. CONCLUSION: The microencapsulation of 3-BrPA represents a promising step towards achieving the goal of systemically deliverable antiglycolytic tumor therapy. The strong anticancer effects of ß-CD-3-BrPA combined with its favorable toxicity profile suggest that clinical trials, particularly in patients with PDAC, should be considered.

Competitive reaction pathways in vibrationally induced photodissociation of H2SO4.

Vibrationally induced photodissociation of sulfuric acid into H2O + SO3 is investigated based on reactive molecular dynamics (MD) simulations. Multisurface adiabatic reactive MD simulations allow us to follow both, H-transfer and water elimination after excitation of the ν9 OH-stretching mode. Analysis of several thousand trajectories finds that the H2O and SO3 fragments have distinct final state distributions with respect to translational, rotational, and vibrational degrees of freedom. Rotational distributions peak at quantum numbers j ≤ 5 for water and j ≈ 60 for SO3. The final state distributions should be useful in identifying products in forthcoming experiments. Based on the MD trajectories, a kinetic scheme has been developed which is able to explain most of the trajectory data and suggests that IVR is very rapid. Typical lifetimes of the excited complex range from several 10 picoseconds to hundreds of nanoseconds, depending on the excitation level. Including temperature and pressure profiles characteristic for the stratosphere in the kinetic model shows that excitations higher than ν9 = 4 can significantly contribute to the photolysis rate. This extends and specifies earlier work in that multi-level modeling is required to understand the significance of vibrationally induced decomposition pathways of sulfuric acid in the middle atmosphere.

Radiosensitization of Pancreatic Cancer Cells In Vitro and In Vivo through Poly (ADP-ribose) Polymerase Inhibition with ABT-888.

OBJECTIVES: To determine whether poly (ADP-ribose) polymerase-1/2 (PARP-1/2) inhibition enhances radiation-induced cytotoxicity of pancreatic adenocarcinoma in vitro and in vivo, and the mechanism by which this occurs. METHODS: Pancreatic carcinoma cells were treated with ABT-888, radiation, or both. In vitro cell viability, apoptosis, and PARP activity were measured. Orthotopic xenografts were generated in athymic mice and treated with ABT-888 (25mg/kg), radiation (5Gy), both, or no treatment. Mice were monitored with bioluminescence imaging. RESULTS: In vitro, treatment with ABT-888 and radiation led to higher rates of cell death after 8days (P < .01). Co-treatment with 5Gy and 1, 10 or 100µmol/l of ABT-888 led to dose enhancement factors of 1.29, 1.41 and 2.36, respectively. Caspase activity was not significantly increased when treated with ABT-888 (10 µmol/l) alone (1.28-fold, P = .08), but became significant when radiation was added (2.03-fold, P < .01). PARP activity increased post-radiation and was abrogated following co-treatment with ABT-888. In vivo, treatment with ABT-888, radiation or both led to tumor growth inhibition (TGI) of 8, 30 and 39days, and survival at 60days of 0%, 0% and 40%, respectively. CONCLUSIONS: ABT-888 with radiation significantly enhanced tumor response in vitro and in vivo. ABT-888 inhibited PAR protein polymerization resulting in dose-dependent feedback up-regulation of PARP and p-ATM suggesting increased DNA damage. This translated into enhancement in TGI and survival with radiation in vivo. In vitro PAR levels correlated with levels of tumor apoptosis suggesting potential as a predictive biomarker. These data are being used to support a Phase I study in locally advanced pancreatic cancer.

Multisurface Adiabatic Reactive Molecular Dynamics.

Adiabatic reactive molecular dynamics (ARMD) simulation method is a surface-crossing algorithm for modeling chemical reactions in classical molecular dynamics simulations using empirical force fields. As the ARMD Hamiltonian is time dependent during crossing, it allows only approximate energy conservation. In the current work, the range of applicability of conventional ARMD is explored, and a new multisurface ARMD (MS-ARMD) method is presented, implemented in CHARMM and applied to the vibrationally induced photodissociation of sulfuric acid (H2SO4) in the gas phase. For this, an accurate global potential energy surface (PES) involving 12 H2SO4 and 4 H2O + SO3 force fields fitted to MP2/6-311G++(2d,2p) reference energies is employed. The MS-ARMD simulations conserve total energy and feature both intramolecular H-transfer reactions and water elimination. An analytical treatment of the dynamics in the crossing region finds that conventional ARMD can approximately conserve total energy for limiting cases. In one of them, the reduced mass of the system is large, which often occurs for simulations of solvated biomolecular systems. On the other hand, MS-ARMD is a general approach for modeling chemical reactions including gas-phase, homogeneous, heterogeneous, and enzymatic catalytic reactions while conserving total energy in atomistic simulations.

Accuracy of off-line bioluminescence imaging to localize targets in preclinical radiation research.

In this study, we investigated the accuracy of using off-line bioluminescence imaging (BLI) and tomography (BLT) to guide irradiation of small soft tissue targets on a small animal radiation research platform (SARRP) with on-board cone beam CT (CBCT) capability. A small glass bulb containing BL cells was implanted as a BL source in the abdomen of 11 mouse carcasses. Bioluminescence imaging and tomography were acquired for each carcass. Six carcasses were setup visually without immobilization and 5 were restrained in position with tape. All carcasses were setup in treatment position on the SARRP where the centroid position of the bulb on CBCT was taken as "truth". In the 2D visual setup, the carcass was setup by aligning the point of brightest luminescence with the vertical beam axis. In the CBCT assisted setup, the pose of the carcass on CBCT was aligned with that on the 2D BL image for setup. For both 2D setup methods, the offset of the bulb centroid on CBCT from the vertical beam axis was measured. In the BLT-CBCT fusion method, the 3D torso on BLT and CBCT was registered and the 3D offset of the respective source centroids was calculated. The setup results were independent of the carcass being immobilized or not due to the onset of rigor mortis. The 2D offset of the perceived BL source position from the CBCT bulb position was 2.3 mm ± 1.3 mm. The 3D offset between BLT and CBCT was 1.5 mm ± 0.9 mm. Given the rigidity of the carcasses, the setup results represent the best that can be achieved with off-line 2D BLI and 3D BLT. The setup uncertainty would require the use of undesirably large margin of 4-5 mm. The results compel the implementation of on-board BLT capability on the SARRP to eliminate setup error and to improve BLT accuracy.

Accuracy of Off-Line Bioluminescence Imaging to Localize Targets in Preclinical Radiation Research.

In this study, we investigated the accuracy of using off-line bioluminescence imaging (BLI) and tomography (BLT) to guide irradiation of small soft tissue targets on a small animal radiation research platform (SARRP) with on-board cone beam CT (CBCT) capability. A small glass bulb containing BL cells was implanted as a BL source in the abdomen of 11 mouse carcasses. Bioluminescence imaging and tomography were acquired for each carcass. Six carcasses were setup visually without immobilization and 5 were restrained in position with tape. All carcasses were setup in treatment position on the SARRP where the centroid position of the bulb on CBCT was taken as "truth". In the 2D visual setup, the carcass was setup by aligning the point of brightest luminescence with the vertical beam axis. In the CBCT assisted setup, the pose of the carcass on CBCT was aligned with that on the 2D BL image for setup. For both 2D setup methods, the offset of the bulb centroid on CBCT from the vertical beam axis was measured. In the BLT-CBCT fusion method, the 3D torso on BLT and CBCT was registered and the 3D offset of the respective source centroids was calculated. The setup results were independent of the carcass being immobilized or not due to the onset of rigor mortis. The 2D offset of the perceived BL source position from the CBCT bulb position was 2.3 mm ± 1.3 mm. The 3D offset between BLT and CBCT was 1.5 mm ± 0.9 mm. Given the rigidity of the carcasses, the setup results represent the best that can be achieved with off-line 2D BLI and 3D BLT. The setup uncertainty would require the use of undesirably large margin of 4-5 mm. The results compel the implementation of on-board BLT capability on the SARRP to eliminate setup error and to improve BLT accuracy.

Subventricular zone localized irradiation affects the generation of proliferating neural precursor cells and the migration of neuroblasts.

Radiation therapy is a part of the standard treatment for brain tumor patients, often resulting in irreversible neuropsychological deficits. These deficits may be due to permanent damage to the neural stem cell (NSC) niche, damage to local neural progenitors, or neurotoxicity. Using a computed tomography-guided localized radiation technique, we studied the effects of radiation on NSC proliferation and neuroblast migration in the mouse brain. Localized irradiation of the subventricular zone (SVZ) eliminated the proliferating neural precursor cells and migrating neuroblasts. After irradiation, type B cells in the SVZ lacked the ability to generate migrating neuroblasts. Neuroblasts from the unirradiated posterior SVZ did not follow their normal migratory path through the irradiated anterior SVZ. Our results indicate that the migrating neuroblasts were not replenished, despite the presence of type B cells in the SVZ post-irradiation. This study provides novel insights into the effects of localized SVZ radiation on neurogenesis and cell migration that may potentially lead to the development of new radiotherapy strategies to minimize damage to NSCs and neuroblast migration.

Development of a novel preclinical pancreatic cancer research model: bioluminescence image-guided focal irradiation and tumor monitoring of orthotopic xenografts.

PURPOSE: We report on a novel preclinical pancreatic cancer research model that uses bioluminescence imaging (BLI)-guided irradiation of orthotopic xenograft tumors, sparing of surrounding normal tissues, and quantitative, noninvasive longitudinal assessment of treatment response. MATERIALS AND METHODS: Luciferase-expressing MiaPaCa-2 pancreatic carcinoma cells were orthotopically injected in nude mice. BLI was compared to pathologic tumor volume, and photon emission was assessed over time. BLI was correlated to positron emission tomography (PET)/computed tomography (CT) to estimate tumor dimensions. BLI and cone-beam CT (CBCT) were used to compare tumor centroid location and estimate setup error. BLI and CBCT fusion was performed to guide irradiation of tumors using the small animal radiation research platform (SARRP). DNA damage was assessed by γ-H2Ax staining. BLI was used to longitudinally monitor treatment response. RESULTS: Bioluminescence predicted tumor volume (R = 0.8984) and increased linearly as a function of time up to a 10-fold increase in tumor burden. BLI correlated with PET/CT and necropsy specimen in size (P < .05). Two-dimensional BLI centroid accuracy was 3.5 mm relative to CBCT. BLI-guided irradiated pancreatic tumors stained positively for γ-H2Ax, whereas surrounding normal tissues were spared. Longitudinal assessment of irradiated tumors with BLI revealed significant tumor growth delay of 20 days relative to controls. CONCLUSIONS: We have successfully applied the SARRP to a bioluminescent, orthotopic preclinical pancreas cancer model to noninvasively: 1) allow the identification of tumor burden before therapy, 2) facilitate image-guided focal radiation therapy, and 3) allow normalization of tumor burden and longitudinal assessment of treatment response.