Sphingosylphosphorylcholine alleviates pressure overload-induced myocardial remodeling in mice via inhibiting CaM-JNK/p38 signaling pathway.

Apoptosis plays a critical role in the development of heart failure, and sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid naturally occurring in blood plasma. Some studies have shown that SPC inhibits hypoxia-induced apoptosis in myofibroblasts, the crucial non-muscle cells in the heart. Calmodulin (CaM) is a known SPC receptor. In this study we investigated the role of CaM in cardiomyocyte apoptosis in heart failure and the associated signaling pathways. Pressure overload was induced in mice by trans-aortic constriction (TAC) surgery. TAC mice were administered SPC (10 µM·kg-1·d-1) for 4 weeks post-surgery. We showed that SPC administration significantly improved survival rate and cardiac hypertrophy, and inhibited cardiac fibrosis in TAC mice. In neonatal mouse cardiomyocytes, treatment with SPC (10 µM) significantly inhibited Ang II-induced cardiomyocyte hypertrophy, fibroblast-to-myofibroblast transition and cell apoptosis accompanied by reduced Bax and phosphorylation levels of CaM, JNK and p38, as well as upregulated Bcl-2, a cardiomyocyte-protective protein. Thapsigargin (TG) could enhance CaM functions by increasing Ca2+ levels in cytoplasm. TG (3 µM) annulled the protective effect of SPC against Ang II-induced cardiomyocyte apoptosis. Furthermore, we demonstrated that SPC-mediated inhibition of cardiomyocyte apoptosis involved the regulation of p38 and JNK phosphorylation, which was downstream of CaM. These results offer new evidence for SPC regulation of cardiomyocyte apoptosis, potentially providing a new therapeutic target for cardiac remodeling following stress overload.

Inhibition of Erb-B2 Receptor Tyrosine Kinase 3 and Associated Regulatory Pathways Potently Impairs Malignant Peripheral Nerve Sheath Tumor Proliferation and Survival.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, currently untreatable Schwann cell-derived neoplasms with hyperactive mitogen-activated protein kinase and mammalian target of rapamycin signaling pathways. To identify potential therapeutic targets, previous studies used genome-scale shRNA screens that implicated the neuregulin-1 receptor erb-B2 receptor tyrosine kinase 3 (erbB3) in MPNST proliferation and/or survival. The current study shows that erbB3 is commonly expressed in MPNSTs and MPNST cell lines and that erbB3 knockdown inhibits MPNST proliferation and survival. Kinomic and microarray analyses of Schwann and MPNST cells implicate Src- and erbB3-mediated calmodulin-regulated signaling as key pathways. Consistent with this, inhibition of upstream (canertinib, sapitinib, saracatinib, and calmodulin) and parallel (AZD1208) signaling pathways involving mitogen-activated protein kinase and mammalian target of rapamycin reduced MPNST proliferation and survival. ErbB inhibitors (canertinib and sapitinib) or erbB3 knockdown in combination with Src (saracatinib), calmodulin [trifluoperazine (TFP)], or proviral integration site of Moloney murine leukemia kinase (AZD1208) inhibition even more effectively reduces proliferation and survival. Drug inhibition enhances an unstudied calmodulin-dependent protein kinase IIα phosphorylation site in an Src-dependent manner. The Src family kinase inhibitor saracatinib reduces both basal and TFP-induced erbB3 and calmodulin-dependent protein kinase IIα phosphorylation. Src inhibition (saracatinib), like erbB3 knockdown, prevents these phosphorylation events; and when combined with TFP, it even more effectively reduces proliferation and survival compared with monotherapy. These findings implicate erbB3, calmodulin, proviral integration site of Moloney murine leukemia kinases, and Src family members as important therapeutic targets in MPNSTs and demonstrate that combinatorial therapies targeting critical MPNST signaling pathways are more effective.

Knockdown of CALM2 increases the sensitivity to afatinib in HER2-amplified gastric cancer cells by regulating the Akt/FoxO3a/Puma axis.

Gastric cancer (GC) is a global health issue that lacks effective treatment options. Afatinib is a tyrosine kinase inhibitor (TKI) that has shown promising results in the treatment of GC. However, resistance to afatinib is inevitable and hampers its clinical application. To date, there is limited knowledge regarding the mechanisms underlying the resistance of GC cells to afatinib. This study aimed to identify novel factors that may contribute to the resistance of GC cells to afatinib. We found that upregulation of calmodulin 2 (CALM2), a member of the CALM family, confers resistance to afatinib in GC cells. Knockdown of CALM2 can overcome the resistance to afatinib by promoting mitochondrial apoptosis in a caspase-dependent manner. Mechanistically, it was found that the downregulation of CALM2 led to the upregulation of the FoxO3a/Puma axis. Inhibition of either FoxO3a or Puma abrogated the effects of CALM2 downregulation in GC cells. In addition, we revealed that CALM2 knockdown inhibited Akt signaling, which is responsible for blocking the FoxO3a/Puma axis. Altogether, our results indicated that CALM2 could be considered a potential target to overcome the resistance of GC cells to afatinib.

Hemoglobin substitutes.

Orthopaedic patients frequently require blood transfusions to treat peri-operative anemia. Research in the area of hemoglobin substitutes has been of great interest since it holds the promise of reducing the reliance on allogeneic blood transfusions. The three categories of hemoglobin substitutes are (1) cell-free, extracellular hemoglobin preparations made from human or bovine hemoglobin (hemoglobin-based oxygen carriers or HBOCs); (2) fluorine-substituted linear or cyclic carbon chains with a high oxygen-carrying capacity (perfluorocarbons); and (3) liposome-encapsulated hemoglobin. Of the three, HBOCs have been the most extensively studied and tested in preclinical and clinical trials that have shown success in diminishing the number of blood transfusions as well as an overall favorable side-effect profile. This has been demonstrated in vascular, cardiothoracic, and orthopaedic patients. HBOC-201, which is a preparation of cell-free bovine hemoglobin, has been approved for clinical use in South Africa. These products may well become an important tool for physicians treating peri-operative anemia in orthopaedic patients.

Potentiation of vincristine effect in human and murine gliomas by calcium channel blockers or calmodulin inhibitors.

The effects of calcium channel blockers and calmodulin inhibitors on vincristine cytotoxicity were studied in vitro with five glioma cell lines: three human glioblastomas, one rat glioma, and one mouse ependymoblastoma. One human glioblastoma and the rat glioma were resistant to vincristine in contrast to other glioma cells. The resistance to vincristine was considerably decreased by nontoxic or marginally toxic concentrations of calcium channel blockers or calmodulin inhibitors, although the former was more effective than the latter. In the presence of verapamil, the vincristine cytotoxicity, as measured by cell doubling times, increased 90- and 84-fold in the vincristine-resistant human glioblastoma and rat glioma, respectively. The decrease in the resistance to vincristine was related to a marked increase in the intracellular level of that drug, probably mediated by inhibiting its outward transport. The in vivo studies showed that verapamil or nicardipine administered daily with vincristine for 10 days significantly enhanced the chemotherapeutic effect of vincristine in an intracranially transplanted rat glioma model. An approximately 32% to 118% increase in life span occurred with 15 mg/kg/day of verapamil, depending on the doses of vincristine.

Effects of extracellular calmodulin and calmodulin antagonists on B16 melanoma cell growth.

Two drugs known to inhibit the action of calmodulin, prochlorperazine offP) and N-(6-aminohexyl)-5-chloro-1-napthalene sulfonamide (W7), were investigated for their ability to control cell proliferation in murine B16 melanoma cells in culture. PCP and W7 inhibited [3H]thymidine uptake in these cells, 50% inhibition occurring with 13 microM PCP and 40 microM W7. In the presence of relatively high concentrations of fetal calf serum (FCS), cells withstood high concentrations of both drugs (100 microM PCP and 200 microM W7) and showed increased pigment production. Drug-inhibited DNA synthesis could be reversed by the addition of fresh medium containing FCS or by the addition of exogenous pure calmodulin. Extracellular calmodulin itself stimulated DNA synthesis. FCS was found to contain calmodulin-like activity at concentrations that may be relevant to the stimulation of [3H]thymidine uptake by cells in culture.