Inhibition of metastatic brain cancer in Sonic Hedgehog medulloblastoma using caged nitric oxide albumin nanoparticles.

Medulloblastoma is a tumor of the cerebellum that metastasizes to the leptomeninges of the central nervous system (CNS), including to forebrain and to spinal cord. The inhibitory effect of polynitroxylated albumin (PNA), a caged nitroxide nanoparticle, on leptomeningeal dissemination and metastatic tumor growth was studied in a Sonic Hedgehog transgenic mouse model. PNA treated mice showed an increased lifespan with a mean survival of 95 days (n = 6, P<0.05) compared with 71 days in controls. In primary tumors, proliferation was significantly reduced and differentiation was significantly increased (P<0.001) as shown by Ki-67+ and NeuN+ immunohistochemistry, while cells in spinal cord tumors appeared unaffected. Yet, histochemical analysis of metastatic tumor in spinal cord showed that the mean total number of cells in spinal cord was significantly reduced in mice treated with PNA compared to albumin vehicle (P<0.05). Examination of various levels of the spinal cord showed that PNA treated mice had significantly reduced metastatic cell density in the thoracic, lumbar and sacral spinal cord levels (P<0.05), while cell density in the cervical region was not significantly changed. The mechanism by which PNA may exert these effects on CNS tumors is discussed.

Polynitroxylated PEGylated hemoglobin protects pig brain neocortical gray and white matter after traumatic brain injury and hemorrhagic shock.

Polynitroxylated PEGylated hemoglobin (PNPH, aka SanFlow) possesses superoxide dismutase/catalase mimetic activities that may directly protect the brain from oxidative stress. Stabilization of PNPH with bound carbon monoxide prevents methemoglobin formation during storage and permits it to serve as an anti-inflammatory carbon monoxide donor. We determined whether small volume transfusion of hyperoncotic PNPH is neuroprotective in a porcine model of traumatic brain injury (TBI) with and without accompanying hemorrhagic shock (HS). TBI was produced by controlled cortical impact over the frontal lobe of anesthetized juvenile pigs. Hemorrhagic shock was induced starting 5 min after TBI by 30 ml/kg blood withdrawal. At 120 min after TBI, pigs were resuscitated with 60 ml/kg lactated Ringer's (LR) or 10 or 20 ml/kg PNPH. Mean arterial pressure recovered to approximately 100 mmHg in all groups. A significant amount of PNPH was retained in the plasma over the first day of recovery. At 4 days of recovery in the LR-resuscitated group, the volume of frontal lobe subcortical white matter ipsilateral to the injury was 26.2 ± 7.6% smaller than homotypic contralateral volume, whereas this white matter loss was only 8.6 ± 12.0% with 20-ml/kg PNPH resuscitation. Amyloid precursor protein punctate accumulation, a marker of axonopathy, increased in ipsilateral subcortical white matter by 132 ± 71% after LR resuscitation, whereas the changes after 10 ml/kg (36 ± 41%) and 20 ml/kg (26 ± 15%) PNPH resuscitation were not significantly different from controls. The number of cortical neuron long dendrites enriched in microtubules (length >50 microns) decreased in neocortex by 41 ± 24% after LR resuscitation but was not significantly changed after PNPH resuscitation. The perilesion microglia density increased by 45 ± 24% after LR resuscitation but was unchanged after 20 ml/kg PNPH resuscitation (4 ± 18%). Furthermore, the number with an activated morphology was attenuated by 30 ± 10%. In TBI pigs without HS followed 2 h later by infusion of 10 ml/kg LR or PNPH, PNPH remained neuroprotective. These results in a gyrencephalic brain show that resuscitation from TBI + HS with PNPH protects neocortical gray matter, including dendritic microstructure, and white matter axons and myelin. This neuroprotective effect persists with TBI alone, indicating brain-targeting benefits independent of blood pressure restoration.

Use of Antimetastatic SOD3-Mimetic Albumin as a Primer in Triple Negative Breast Cancer.

Of the deaths attributed to cancer, 90% are due to metastasis. Treatments that prevent or cure metastasis remain elusive. Low expression of extracellular superoxide dismutase (EcSOD or SOD3) has been associated with poor outcomes and increased metastatic potential in multiple types of cancer. Here, we characterize the antimetastatic therapeutic mechanisms of a macromolecular extracellular SOD3-mimetic polynitroxyl albumin (PNA, also known as VACNO). PNA is macromolecular human serum albumin conjugated with multiple nitroxide groups and acts as an SOD-mimetic. Here we show that PNA works as a SOD3-mimetic in a highly metastatic 4T1 mouse model of triple negative breast cancer (TNBC). In vitro, PNA dose dependently inhibited 4T1 proliferation, colony formation, and reactive oxygen species (ROS) formation. In vivo, PNA enhanced reperfusion time in the hypoxic cores of 4T1 tumors as measured by ultrasound imaging. Furthermore, PNA enhanced ultrasound signal intensity within the cores of the 4T1 tumors, indicating PNA can increase blood flow and blood volume within the hypoxic cores of tumors. Lung metastasis from 4T1 flank tumor was inhibited by PNA in the presence or absence of doxorubicin, a chemotherapy agent that produces superoxide and promotes metastasis. In a separate study, PNA increased the survival of mice with 4T1 flank tumors when used in conjunction with three standard chemotherapy drugs (paclitaxel, doxorubicin, and cyclophosphamide), as compared to treatment with chemotherapy alone. In this study, PNA-increased survival was also correlated with reduction of lung metastasis. These results support the hypothesis that PNA works through the inhibition of extracellular superoxide/ROS production leading to the conversion of 4T1 cells from a metastatic tumorigenic state to a cytostatic state. These findings support future clinical trials of PNA as an antimetastatic SOD3-mimetic drug to increase overall survival in TNBC patients.

Automated screening of neurite outgrowth.

Outgrowth of neurites in culture is used for assessing neurotrophic activity. Neurite measurements have been performed very slowly using manual methods or more efficiently with interactive image analysis systems. In contrast, medium-throughput and noninteractive image analysis of neurite screens has not been well described. The authors report the performance of an automated image acquisition and analysis system (IN Cell Analyzer 1000) in the neurite assay. Neuro-2a (N2a) cells were plated in 96-well plates and were exposed to 6 conditions of retinoic acid. Immunofluorescence labeling of the cytoskeleton was used to detect neurites and cell bodies. Acquisition of the images was automatic. The image set was then analyzed by both manual tracing and automated algorithms. On 5 relevant parameters (number of neurites, neurite length, total cell area, number of cells, neurite length per cell), the authors did not observe a difference between the automated analysis and the manual analysis done by tracing. These data suggest that the automated system addresses the same biology as human scorers and with the same measurement precision for treatment effects. However, throughput of the automated system is orders of magnitude higher than with manual methods.