The Allosteric Hemoglobin Effector ITPP Inhibits Metastatic Colon Cancer in Mice.

OBJECTIVE: To test the effects of enhanced intracellular oxygen contents on the metastatic potential of colon cancer. BACKGROUND: Colorectal cancer is the commonest gastrointestinal carcinoma. Distant metastases occur in half of patients and are responsible for most cancer-related deaths. Tumor hypoxia is central to the pathogenesis of metastases. Myo-Inositoltrispyrophosphate (ITPP), a nontoxic, antihypoxic compound, has recently shown significant benefits in experimental cancer, particularly when combined with standard chemotherapy. Whether ITPP protects from distant metastases in primary colon cancer is unknown. METHODS: ITPP alone or combined with FOLFOX was tested in a mouse model with cecal implantation of green fluorescent protein-labeled syngeneic colorectal cancer cells. Tumor development was monitored through longitudinal magnetic resonance imaging-based morphometric analysis and survival. Established serum markers of tumor spread were measured serially and circulating tumor cells were detected via fluorescence measurements. RESULTS: ITPP significantly reduced the occurrence of metastases as well as other indicators of tumor aggressiveness. Less circulating tumor cells along with reduction in malignant serum markers (osteopontin, Cxcl12) were noted. The ITPP benefits also affected the primary cancer site. Importantly, animals treated with ITPP had a significant survival benefit compared with respective controls, while a combination of FOLFOX with ITPP conferred the maximum benefits, including dramatic improvements in survival (mean 86 vs 188 d). CONCLUSIONS: Restoring oxygen in metastatic colon cancer through ITPP inhibits tumor spread and markedly improves animal survival; an effect that is enhanced through the application of subsequent chemotherapy. These promising novel findings call for a clinical trial on ITPP in patients with colorectal cancer, which is under way.

Antihypoxic Potentiation of Standard Therapy for Experimental Colorectal Liver Metastasis through Myo-Inositol Trispyrophosphate.

PURPOSE: Tumor hypoxia activates hypoxia-inducible factors (Hifs), which induce a range of malignant changes including vascular abnormalities. Here, we determine whether inhibition of the hypoxic tumor response through myo-inositol trispyrophosphate (ITPP), a compound with antihypoxic properties, is able to cause prolonged vascular normalization that can be exploited to improve standard-of-care treatment. EXPERIMENTAL DESIGN: We tested ITPP on two syngeneic orthotopic mouse models of lethal colorectal cancer liver metastasis. Tumors were monitored by MRI and analyzed for the hypoxic response and their malignant potential. A Hif activator and in vitro assays were used to define the working mode of ITPP. Hypoxic response and vasculature were re-evaluated 4 weeks after treatment. Finally, we determined survival following ITPP monotherapy, FOLFOX monotherapy, FOLFOX plus Vegf antibody, and FOLFOX plus ITPP, both overlapping and sequential. RESULTS: ITPP reduced tumor load, efficiently inhibited the hypoxic response, and improved survival. These effects were lost when mice were pretreated with a Hif activator. Its immediate effects on the hypoxic response, including an apparent normalization of tumor vasculature, persisted for at least 4 weeks after treatment cessation. Compared with FOLFOX alone, Vegf antibody combined with FOLFOX prolonged survival by <30%, whereas ITPP combined with FOLFOX extended survival by >140%, regardless of whether FOLFOX was given in overlap or after ITPP exposure. CONCLUSIONS: Our findings reveal a truly antihypoxic mechanism for ITPP and demonstrate the capacity of this nontoxic compound to potentiate the efficacy of existing anticancer treatment in a way amenable to clinical translation. Clin Cancer Res; 22(23); 5887-97. ©2016 AACR.

Fullerenols and glucosamine fullerenes reduce infarct volume and cerebral inflammation after ischemic stroke in normotensive and hypertensive rats.

Cerebral inflammation plays a crucial role in the pathophysiology of ischemic stroke and is involved in all stages of the ischemic cascade. Fullerene derivatives, such as fullerenol (OH-F) are radical scavengers acting as neuroprotective agents while glucosamine (GlcN) attenuates cerebral inflammation after stroke. We created novel glucosamine-fullerene conjugates (GlcN-F) to combine their protective effects and compared them to OH-F regarding stroke-induced cerebral inflammation and cellular damage. Fullerene derivatives or vehicle was administered intravenously in normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) immediately after transient middle cerebral artery occlusion (tMCAO). Infarct size was determined at day 5 and neurological outcome at days 1 and 5 after tMCAO. CD68- and NeuN-staining were performed to determine immunoreactivity and neuronal survival respectively. Cytokine and toll like receptor 4 (TLR-4) expression was assessed using quantitative real-time PCR. Magnetic resonance imaging revealed a significant reduction of infarct volume in both, WKY and SHR that were treated with fullerene derivatives. Treated rats showed an amelioration of neurological symptoms as both OH-F and GlcN-F prevented neuronal loss in the perilesional area. Cerebral immunoreactivity was reduced in treated WKY and SHR. Expression of IL-1ß and TLR-4 was attenuated in OH-F-treated WKY rats. In conclusion, OH-F and GlcN-F lead to a reduction of cellular damage and inflammation after stroke, rendering these compounds attractive therapeutics for stroke.

Systemic protection through remote ischemic preconditioning is spread by platelet-dependent signaling in mice.

UNLABELLED: Remote ischemic preconditioning (RIPC), the repetitive transient mechanical obstruction of vessels at a limb remote to the operative site, is a novel strategy to mitigate distant organ injury associated with surgery. In the clinic, RIPC has demonstrated efficacy in protecting various organs against ischemia reperfusion (IR), but a common mechanism underlying the systemic protection has not been identified. Here, we reasoned that protection may rely on adaptive physiological responses toward local stress, as is incurred through RIPC. Standardized mouse models of partial hepatic IR and of RIPC to the femoral vascular bundle were applied. The roles of platelets, peripheral serotonin, and circulating vascular endothelial growth factor (Vegf) were studied in thrombocytopenic mice, Tph1(-) (/) (-) mice, and through neutralizing antibodies, respectively. Models of interleukin-10 (Il10) and matrix metalloproteinase 8 (Mmp8) deficiency were used to assess downstream effectors of organ protection. The protection against hepatic IR through RIPC was dependent on platelet-derived serotonin. Downstream of serotonin, systemic protection was spread through up-regulation of circulating Vegf. Both RIPC and serotonin-Vegf induced differential gene expression in target organs, with Il10 and Mmp8 displaying consistent up-regulation across all organs investigated. Concerted inhibition of both molecules abolished the protective effects of RIPC. RIPC was able to mitigate pancreatitis, indicating that it can protect beyond ischemic insults. CONCLUSIONS: We have identified a platelet-serotonin-Vegf-Il10/Mmp8 axis that mediates the protective effects of RIPC. The systemic action, the conservation of RIPC effects among mice and humans, and the protection beyond ischemic insults suggest that the platelet-dependent axis has evolved as a preemptive response to local stress, priming the body against impending harm.

Deoxysphingolipids, novel biomarkers for type 2 diabetes, are cytotoxic for insulin-producing cells.

Irreversible failure of pancreatic ß-cells is the main culprit in the pathophysiology of diabetes, a disease that is now a global epidemic. Recently, elevated plasma levels of deoxysphingolipids, including 1-deoxysphinganine, have been identified as a novel biomarker for the disease. In this study, we analyzed whether deoxysphingolipids directly compromise the functionality of insulin-producing Ins-1 cells and primary islets. Treatment with 1-deoxysphinganine induced dose-dependent cytotoxicity with senescent, necrotic, and apoptotic characteristics and compromised glucose-stimulated insulin secretion. In addition, 1-deoxysphinganine altered cytoskeleton dynamics, resulting in intracellular accumulation of filamentous actin and activation of the Rho family GTPase Rac1. Moreover, 1-deoxysphinganine selectively upregulated ceramide synthase 5 expression and was converted to 1-deoxy-dihydroceramides without altering normal ceramide levels. Inhibition of intracellular 1-deoxysphinganine trafficking and ceramide synthesis improved the viability of the cells, indicating that the intracellular metabolites of 1-deoxysphinganine contribute to its cytotoxicity. Analyses of signaling pathways identified Jun N-terminal kinase and p38 mitogen-activated protein kinase as antagonistic effectors of cellular senescence. The results revealed that 1-deoxysphinganine is a cytotoxic lipid for insulin-producing cells, suggesting that the increased levels of this sphingolipid observed in diabetic patients may contribute to the reduced functionality of pancreatic ß-cells. Thus, targeting deoxysphingolipid synthesis may complement the currently available therapies for diabetes.

Activation of mitogen-activated protein kinases during granulocyte apoptosis in patients with severe sepsis.

Reduction of neutrophil apoptosis represents a major cause for granulocytosis and increases the destructive potential of theses cells during systemic inflammatory response syndrome (SIRS) and sepsis. In this light, the role of protein kinases for the regulation of altered neutrophil apoptosis under infectious conditions was investigated. Neutrophils, obtained from patients with severe sepsis (n = 18), were incubated ex vivowith either LPS (1 microg/mL) or interferon-gamma (IFN-gamma; 10 ng/mL) for 16 h. Apoptosis was determined by propidium iodine (PI) staining of DNA fragments and was compared with the rate of spontaneous apoptosis. Tyrosine kinases were inhibited by herbimycin (1 microM), the mitogen-activated protein (MAP) kinase ERK was inhibited with PD98059 (50 microM), and p38 MAP kinase was inhibited with SB203580 (5 microM). Herbimycin reconstituted LPS-reduced apoptosis in neutrophils from controls (39.9 +/- 3.8%) and patients (20.8 +/- 2.8%) to levels seen in spontaneous apoptosis (70.9 +/- 2.8% and 40.7 +/- 3.7%, respectively). Inhibition of the ERK kinase yielded similar results, whereas SB203580 had no effect on LPS-reduced apoptosis. However, inhibition of p38 partially reconstituted IFN-gamma-reduced apoptosis (51.3 +/- 7.7% and 25.6 +/- 5.8%) and increased spontaneous apoptosis (82.4 +/- 3.3% and 42.0 +/- 5.8%) in controls and patients, respectively. Western blot analysis revealed phosphorylation of both MAP kinases by LPS, but not by IFN-gamma. Inhibition of MAP kinases did not augment neutrophil apoptosis in patients to the level seen in controls, indicating that other mechanisms must be involved in the regulation of neutrophil apoptosis. Although the ERK kinase regulates LPS-induced reduction of apoptosis, the p38 MAP kinase might be involved in IFN-gamma signaling and the feedback regulation of neutrophil apoptosis.