Upon activation, mTOR, a serine/threonine kinase, is responsible for phosphorylation and activation of its two major downstream components, p70S6K and eIF4E-binding protein 1 (4E-BP1)

Upon activation, mTOR, a serine/threonine kinase, is responsible for phosphorylation and activation of its two major downstream components, p70S6K and eIF4E-binding protein 1 (4E-BP1). of Akt, p70S6K, and eukaryotic initiation factor 4E binding protein 1 included in mammalian target of rapamycin (mTOR) pathways, which are located downstream of Akt. Furthermore, rapamycin not only drastically inhibited CXCL16-induced PCa cell invasion and growth but reduced secretion of IL-8 or VEGF levels and inhibited expression of other CXCR6 targets including CD44 and matrix metalloproteinase 3 in PCa cells. Together, our data shows for the first time that the CXCR6/AKT/mTOR pathway plays a central role in the development of PCa. Blocking the CXCR6/AKT/mTOR signaling pathway may prove beneficial to prevent metastasis and provide a more effective therapeutic strategy for PCa. L-Citrulline Introduction The high mortality rate of prostate cancer (PCa) is closely associated with the spread of malignant cells to various tissues in the body including bone. Intriguingly, evidence suggests that chemokines are likely to play a crucial role in mediating the continuous series of events leading to tumor growth and metastasis (1C3). Therefore, it is important to understand chemokine signaling in PCa to get a better understanding of the underlying mechanisms leading to tumor invasion and metastasis, and to develop prognostic and therapeutic strategies in this disease. During the events in which PCa metastasizes to bone marrow, the cancer cells must first migrate out of the prostate, adhere to the endothelial marrow cells, and subsequently extravasate and proliferate in the extravascular marrow spaces. All of these events are functionally similar to hematopoietic cells as they move in and out of the bone marrow, an event that seems to be largely regulated by chemokines (3, 4). Chemokines are small proinflammatory chemoattractant cytokines that bind to G-proteinCcoupled seven-span transmembrane receptors that are major regulators of cellular trafficking. Based on the number and position of conserved cysteine residues near the NH2 terminus, chemokines are classified by their structure into four groups, namely CC, CXC, C, and CX3C. Over the past several years, there is emerging evidence that multiple pairs of chemokines and their receptors play critical roles in cancer progression (5C7). Our group and others have shown that CXCL12 and its receptor are critical elements in growth and metastasis of PCa to tissues that produce large quantities of CXCL12 (8, 9). More recently, we have shown that a second CXCL12 receptor, CXCR7/RDC1, also plays a central role in PCa metastasis and progression (10). However, blockade of the CXCR4 or CXCR7 receptor only partially blocks metastatic behavior L-Citrulline from each well transferred into 96 wells and read on a multiwell scanning spectrophotometer (Molecular Devices Corp.) at OD450. Flow cytometry For surface chemokine receptor detection, 2 105 cells were incubated at 2C to 8C for 40 min with nonspecific isotype-matched controls, mouse to human IgG (BD Biosciences), and 20 g/mL of each of the following murine monoclonal antibodies: anti-human CXCR6 (R&D Systems; FAB699P). The mouse primary antibodies were then detected by incubating the cells at 25 C for 45 min with phycoerythrin. The cells were washed twice with PBS, resuspended and fixed in 1% (w/v) paraformaldehyde for analysis. Ten thousands cells L-Citrulline from each sample were evaluated for fluorescence detection using FACScan (Becton Dickinson), and the data were analyzed with CellQuest software (Becton Dickinson). ELISA Antibody HNRNPA1L2 sandwich ELISAs were used to evaluate interleukin (IL)-8, and vascular endothelial growth factor (VEGF) levels in the PCa cell CM (R&D Systems) as previously described (22). Angiogenesis antibody arrays The expression of 19 cytokines was evaluated using Angiogenesis antibody arrays (Panomics). The membranes were exposed to blocking buffer for 1 h at 25C, and incubated with CM or control medium up to 2h at 25C. After washing thrice,.