Purification and characterization of platelet aggregation inhibitors from snake venoms

Proteins that inhibit glycoprotein (GP) IIb/IIIa mediated platelet aggregation have been purified from the venom of two snake species. A small platelet aggregation inhibitor (pl.AI), multisquamatin (Mr=5,700), was purified from Echis multisquamatus venom by hydrophobic interaction HPLC and two steps on C18 reverse phase HPLC. A larger pl.AI, contortrostatin (Mr=15,000), was purified by a similar HPLC procedure from the venom of Agkistrodon contortrix contortrix. Both pl.AIs inhibit ADP-induced human, canine and rabbit platelet aggregation using platelet rich plasma (PRP). Multisquamatin has an IC50 of 97 nM, 281 nM and 333 nM for human, canine and rabbit PRP, respectively. Contortrostatin has an IC50 of 49 nM, 120 nM and 1,150 nM for human, canine and rabbit PRP, respectively. In a competitive binding assay using 125I-7E3 (a monoclonal antibody to GPIIb/IIIa that inhibits platelet aggregation) both contortrostatin and multisquamatin demonstrated GPIIb/IIIa specific binding to human and canine platelets. The IC50 for contortrostatin displacement of 7E3 binding to human and canine GPIIb/IIIa is 27 nM and 16 nM, respectively and for multisquamatin it is 3 nM and 63 nM, respectively. Our results indicate that both pl.AIs inhibit platelet aggregation by binding with high affinity to GPIIb/IIIa.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31863/1/0000813.pd

Targeted anti-interleukin-6 monoclonal antibody therapy for cancer: a review of the rationale and clinical evidence.

International audienceInterleukin (IL)-6, a pleiotropic cytokine with varied systemic functions, plays a major role in inflammatory processes. It modulates the transcription of several liver-specific genes during acute inflammatory states, particularly C-reactive protein, and controls the survival of normal plasmablastic cells. In addition, IL-6 has been implicated in hematopoiesis as a cofactor in stem cell amplification and differentiation. This article is the first review of clinical studies in the 1990s with anti-IL-6 monoclonal antibodies (mAbs) in the treatment of patients with cancer and related lymphoproliferative disorders. In six clinical studies of mAbs to IL-6 with BE-8 or CNTO 328 in patients with multiple myeloma, renal cell carcinoma, and B-lymphoproliferative disorders, anti-IL-6 mAb treatment decreased C-reactive protein levels in all patients. In most patients, levels decreased below detectable limits. The antibodies were well tolerated, and no serious adverse effects were observed in the vast majority of studies. The fact that anti-IL-6 mAb therapy decreased the incidence of cancer-related anorexia and cachexia may also be useful in the treatment of cancer patients

MARIZOMIB (NPI-0052) ACTIVITY AS A SINGLE AGENT IN MALIGNANT GLIOMA

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor, which displays innate resistance to multiple treatment modalities. Previous studies have shown that proteasome inhibition can be used as a strategy for treating this malignancy. Marizomib (NPI-0052) is a second generation irreversible proteasome inhibitor, which has a more lipophilic structure and has a broader and more prolonged inhibition profile for 20S proteasome activities compared to bortezomib and carfilzomib. While bortezomib and carfilzomib have only modest activity in gliomas, marizomib might potentially be a novel therapeutic strategy for primary brain tumors. In these studies, we investigated the in vitro activities of marizomib in primary glioma cultures, neural stem/progenitor cells (NSC) and as well as in established human malignant glioma lines. The effect of marizomib on cell proliferation, proteasome activity, motility, apoptosis and Reactive Oxygen Species (ROS) were evaluated in glioma cell lines. The sensitivities varied in function of the pathology of the tumor, with the malignant glioma stem-like cells being the most severely affected, in contrast with the low-grade glioma and NSC-derived cultures. Marizomib inhibited the proliferation of U251-MG and D54-MG cell lines with a half maximal effective concentration (EC50) of 52nM and 20nM respectively, along with a significant decrease in cell migration and invasion. Marizomib treatment of human glioma cells was associated with increased ROS production and apoptosis, along with activation of caspase-3 and cleavage of PARP. Those effects of marizomib can be suppressed by exposure to the ROS quenching agent N-acetyl cysteine (NAC). These preclinical studies demonstrate a significant anti-glioma effect of marizomib. Marizomib has relatively little effect on neural stem/progenitor cells suggesting minimal neurotoxicity. But importantly, unlike bortezomib and carfilzomib, marizomib can cross the blood brain barrier. Additional research into the use of marizomib in malignant gliomas orthotropic models is in progress and will be presented during the meeting

ET-16MARIZOMIB (NPI-0052) ACTIVITY AS A SINGLE AGENT IN MALIGNANT GLIOMA

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor, which displays innate resistance to multiple treatment modalities. Previous studies have shown that proteasome inhibition can be used as a strategy for treating this malignancy. Marizomib (NPI-0052) is a second generation irreversible proteasome inhibitor, which has a more lipophilic structure and has a broader and more prolonged inhibition profile for 20S proteasome activities compared to bortezomib and carfilzomib. While bortezomib and carfilzomib have only modest activity in gliomas, marizomib might potentially be a novel therapeutic strategy for primary brain tumors. In these studies, we investigated the in vitro activities of marizomib in primary glioma cultures, neural stem/progenitor cells (NSC) and as well as in established human malignant glioma lines. The effect of marizomib on cell proliferation, proteasome activity, motility, apoptosis and Reactive Oxygen Species (ROS) were evaluated in glioma cell lines. The sensitivities varied in function of the pathology of the tumor, with the malignant glioma stem-like cells being the most severely affected, in contrast with the low-grade glioma and NSC-derived cultures. Marizomib inhibited the proliferation of U251-MG and D54-MG cell lines with a half maximal effective concentration (EC50) of 52nM and 20nM respectively, along with a significant decrease in cell migration and invasion. Marizomib treatment of human glioma cells was associated with increased ROS production and apoptosis, along with activation of caspase-3 and cleavage of PARP. Those effects of marizomib can be suppressed by exposure to the ROS quenching agent N-acetyl cysteine (NAC). These preclinical studies demonstrate a significant anti-glioma effect of marizomib. Marizomib has relatively little effect on neural stem/progenitor cells suggesting minimal neurotoxicity. But importantly, unlike bortezomib and carfilzomib, marizomib can cross the blood brain barrier. Additional research into the use of marizomib in malignant gliomas orthotropic models is in progress and will be presented during the meeting

Ectopic alpha IIb beta 3 integrin signaling involves 12-lipoxygenase- and PKC-mediated serine phosphorylation events in melanoma cells

Megakaryocytic genes such as alpha IIb beta3 can be expressed by malignant cells as part of the disturbances in their gene regulation. However, the function of the gene product greatly depends on the interaction of the ectopic protein with the new environment. The outside-in signaling of the ectopically expressed alpha IIb beta3 integrin was studied in B16a murine melanoma cells using a monoclonal antibody, specifically directed to the activated conformation of alpha IIb beta3, PAC-1 and the physiological ligand, fibrinogen. Ligation of alpha IIb beta3 induced downregulation of FAK but serine phosphorylation of three protein bands, 20/21, 85 and 140 kDa within 1-15 min. Flow cytometry indicated that the ligation of the receptor in B16a cells induces similar to 50%. increase in phosphoserine positive cells within 5-15 min. 12-lipoxygenase is placed downstream in the signaling pathway, since ligation of alpha IIb beta3 induces 12-HETE production within 5 min and pretreatment of tumor cells with select lipoxygenase inhibitior, Baicalein, prevents the increase in serine phosphorylation. Confocal microscopy of adherent tumor cells demonstrated rearrangement of actin filaments upon alpha IIb beta3 ligation paralleled by downregulation of p125FAK and phoshotyrosine(+) adhesion plaques and translocation of PKC alpha to stress fibers and cortical actin. PKC appears to be the major effector serine kinase of the alpha IIb beta3-coupled signaling pathway, since pretreatment of tumor cells with a select PKC inhibitor, Calphostin C, prevents the ligation-induced serine phosphorylation. Previous studies have indicated a role for the 12-lipoxygenase-PKC signaling pathway in platelet aggregation as well as tumor invasion, therefore the involvement of this cascade in the signaling of the ectopic alpha IIb beta3 integrin may partially explain its role in tumor progression