Novel therapeutic strategies for spinal osteosarcomas.

At the dawn of the third millennium, cancer has become the bane of twenty-first century man, and remains a predominant public health burden, affecting welfare and life expectancy globally. Spinal osteogenic sarcoma, a primary spinal malignant tumor, is a rare and challenging neoplastic disease to treat. After the conventional therapeutic modalities of chemotherapy, radiation and surgery have been exhausted, there is currently no available alternative therapy in managing cases of spinal osteosarcoma. The defining signatures of tumor survival are characterised by cancer cell ability to stonewall immunogenic attrition and apoptosis by various means. Some of these biomarkers, namely immune-checkpoints, have recently been exploited as druggable targets in osteosarcoma and many other different cancers. These promising strides made by the use of reinvigorated immunotherapeutic approaches may lead to significant reduction in spinal osteosarcoma disease burden and corresponding reciprocity in increase of survival rates. In this review, we provide the background to spinal osteosarcoma, and proceed to elaborate on contribution of the complex ecology within tumor microenvironment giving arise to cancerous immune escape, which is currently receiving considerable attention. We follow this section on the tumor microenvironment by a brief history of cancer immunity. Also, we draw on the current knowledge of treatment gained from incidences of osteosarcoma at other locations of the skeleton (long bones of the extremities in close proximity to the metaphyseal growth plates) to make a case for application of immunity-based tools, such as immune-checkpoint inhibitors and vaccines, and draw attention to adverse upshots of immune-checkpoint blockers as well. Finally, we describe the novel biotechnique of CRISPR/Cas9 that will assist in treatment approaches for personalized medication.

Cyclic AMP acts through Rap1 and JNK signaling to increase expression of cutaneous smooth muscle alpha2C-adrenoceptors.

Cold increases cutaneous vasoconstriction by unmasking the contractile activity of alpha(2C)-adrenoceptors (alpha(2C)-ARs) in vascular smooth muscle cells (VSMCs), which is mediated by the cold-induced mobilization of alpha(2C)-ARs from the transGolgi to the cell surface. The expression of alpha(2C)-ARs in human cutaneous VSMCs is under dual regulation by cyclic AMP: gene transcription is inhibited by cyclic AMP acting through protein kinase A but is increased by cyclic AMP acting through the exchange protein directly activated by cyclic AMP (EPAC) and the GTP-binding protein Rap1. Experiments were performed to further characterize the Rap1 signaling pathway. Forskolin (10 muM), the selective EPAC activator, 8-pCPT-2'-O-Me-cyclic AMP (CMC; 100 microM), or a constitutively active mutant of Rap1 (Rap1CA) increased the activity of c-Jun NH(2)-terminal kinase (JNK) in human cutaneous VSMCs. This was associated with the increased phosphorylation of c-Jun and activation of an activator protein (AP)-1 reporter construct, which were inhibited by the JNK inhibitor SP600125 (3 microM). Rap1CA increased the activity of an alpha(2C)-AR promoter-reporter construct, which was inhibited by SP600125 (3 microM) or by the mutation of an AP-1 binding site in the alpha(2C)-AR promoter. Furthermore, forskolin (10 microM) or CMC (100 microM) increased the expression of the alpha(2C)-AR protein, and these effects were inhibited by SP600125 (3 microM). Therefore, cyclic AMP increases the expression of alpha(2C)-ARs in cutaneous VSMCs by activating a novel Rap1 signaling pathway, mediated by the activation of JNK, AP-1, and the subsequent transcriptional activation of the alpha(2C)-AR gene. By increasing the expression of cold-responsive alpha(2C)-ARs, this pathway may contribute to enhanced cold-induced vasoconstriction in the cutaneous circulation, including Raynaud's phenomenon.

Estrogen increases smooth muscle expression of alpha2C-adrenoceptors and cold-induced constriction of cutaneous arteries.

Raynaud's phenomenon, which is characterized by intense cold-induced constriction of cutaneous arteries, is more common in women compared with men. Cold-induced constriction is mediated in part by enhanced activity of alpha(2C)-adrenoceptors (alpha(2C)-ARs) located on vascular smooth muscle cells (VSMs). Experiments were therefore performed to determine whether 17beta-estradiol regulates alpha(2C)-AR expression and function in cutaneous VSMs. 17beta-Estradiol (0.01-10 nmol/l) increased expression of the alpha(2C)-AR protein and the activity of the alpha(2C)-AR gene promoter in human cultured dermal VSMs, which was assessed following transient transfection of the cells with a promoter-reporter construct. The effect of 17beta-estradiol was associated with increased accumulation of cAMP and activation of the cAMP-responsive Rap2 GTP-binding protein. Transient transfection of VSMs with a dominant-negative mutant of Rap2 inhibited the 17beta-estradiol-induced activation of the alpha(2C)-AR gene promoter, whereas a constitutively active mutant of Rap2 increased alpha(2C)-AR promoter activity. The effects of 17beta-estradiol were inhibited by the estrogen receptor (ER) antagonist, ICI-182780 (1 micromol/l), and were mimicked by a cell-impermeable form of the hormone (estrogen:BSA) or by the selective ER-alpha receptor agonist 4,4',4'''-(4-propyl-[(1)H]-pyrazole-1,3,5-triyl)tris-phenol (PPT; 10 nmol/l) or the selective ER-beta receptor agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; 10 nmol/l). Therefore, 17beta-estradiol increased expression of alpha(2C)-ARs by interacting with cell surface receptors to cause a cAMP/Rap2-dependent increase in alpha(2C)-AR transcription. In mouse tail arteries, 17beta-estradiol (10 nmol/l) increased alpha(2C)-AR expression and selectively increased the cold-induced amplification of alpha(2)-AR constriction, which is mediated by alpha(2C)-ARs. An estrogen-dependent increase in expression of cold-sensitive alpha(2C)-ARs may contribute to the increased activity of cold-induced vasoconstriction under estrogen-replete conditions.

Rho kinase mediates cold-induced constriction of cutaneous arteries: role of alpha2C-adrenoceptor translocation.

Cold-induced vasoconstriction in cutaneous blood vessels is mediated in part by increased activity of vascular smooth muscle alpha2-adrenoceptors (VSM alpha2-ARs). In mouse cutaneous arteries, alpha2C-ARs are normally silent at 37 degrees C but mediate cold-induced augmentation of alpha2-AR responsiveness. In transfected HEK293 cells, this functional rescue is mediated by cold-induced translocation of alpha2C-ARs from the Golgi to the plasma membrane. Experiments were performed to determine the role of Rho/Rho kinase signaling in this process. Inhibition of Rho kinase (fasudil, Y27632 or H-1152) did not affect constriction of isolated mouse tail arteries to the alpha2-AR agonist UK 14 304 at 37 degrees C but dramatically reduced the augmented responses to the agonist at 28 degrees C. After Rho kinase inhibition, cooling no longer increased constriction evoked by alpha2-AR stimulation. Cooling (to 28 degrees C) activated Rho in VSM cells and increased the calcium sensitivity of constriction in alpha toxin-permeabilized arteries. Stimulation of alpha2-ARs in VSM cells had no effect on Rho activity or calcium sensitivity at 37 degrees C or 28 degrees C. In HEK293 cells transfected with alpha2C-ARs, cooling (to 28 degrees C) stimulated the translocation of alpha2C-ARs to the plasma membrane and this effect was prevented by inhibition of Rho kinase, using fasudil or RNA interference. Consistent with inhibition of the spatial rescue of alpha2C-ARs, fasudil inhibited alpha2-AR-mediated mobilization of calcium in tail arteries at 28 degrees C but not 37 degrees C. Therefore, cold-induced activation of Rho/Rho kinase can mediate cold-induced constriction in cutaneous arteries by enabling translocation of alpha2C-ARs to the plasma membrane and by increasing the calcium sensitivity of the contractile process.

Accumulation of diflubenzuron in bolti fish Orechromis niloticus.

Orechromis niloticus fingerlings were exposed to the insect growth inhibitor diflubenzuron 1-(2,6-Difluorobenzoyl)3-(4-chlorophenyl)urea for 21 days. Diflubenzuron was introduced to the aquariums where fish were maintained at the beginning of the experiment, then its level in water, gills and liver was detected after 1, 7, 14 and 21 days. The fish accumulated diflubenzuron 76 and 99 times greater than the water content when kept in an ambient concentration of 2.5 and 5 mg/l, respectively, indicating a low bioaccumulation potential. Some degradation products of diflubenzuron were found mainly in liver and water.