AM plays a role in the maintenance of blood circulation and has antithrombotic effects. There are two important factors that regulate blood coagulation which are plasma protease factor VII/VIIa (FVIIa) and tissue factor (TF). Their binding initiates the blood coagulation cascade (). The Tissue Factor Pathway Inhibitor (TFPI) is an inhibitor for TF-FVIIa complex which is secreted and synthesized by endothelial cells where the Heparan Sulfate Proteo Glycan (HSPG) helps TFPI to express at the cell surface. In thrombotic diseases the levels of AM and TFPI are increased, illustrating the possibility of existing correlations between them (). The cationic charge provided by the amino acid of AM is responsible for inducing the release of TFPI from the surface of ES, where TFPI binds to cell surface HSPG via the positively charged C-terminal region of the TFPI sequence. Additionally, in the ECs AM has the ability to induce antithrombin (AT) and urokinase type plasminogen activator (uPA) release (). AM has been shown to be a protection factor for organs in hypertension, oxidative stress, atherosclerosis, ischemia, and sepsis which results in multi organ failure. In rodent models, prolonged infusion or the overexpression of AM prevented thickness of the internal part of the capillary which is called intima by preventing the formation of fatty streaks and perivascular hyperplasia in order to protect the blood vessels. This shows AM is an important regulator for maintaining the circulatory system function (). The administration of AM systemically into the model of tail lymphoedema resulted in an improvement of lymphoedema compared to controls. The blood and lymphatic vessels number were shown to be increased at the site of injury. This finding emphasises the role of AM in angiogenic and lymphogenic processes (). AM has many other biological actions such as increasing cAMP in the cell, the
Western blotting Item Supplier Phosphate Buffered Saline (PBS) - tablets OXOID Tween 20 Fisher Scientific Biotinylated Protein Ladder Detection Cell Signalling Precision Plus Protein Standards Bio Rad Immobilon Transfer Membrane (PVDF membrane) Millipore SuperSignal®West Dura Extended Duration Substrate Thermo Scientific Mini-PROTEAN Tetra Cell BioRad Mini Trans-Blot® Electrophoretic Transfer Cell BioRad Film Hyperfilm™ Amersham Developer AGAFA Fixer GS-900™ Calibrated Densitometer BioRad 1.5 mm cast plates (Bio RAD) Materials used in the experiment Tris/Glycine 10X Item Amount per Litre Supplier Tris 250mM 30.3g Fisher Scientific Glycine 1.92M 144.1g Fisher Scientific Deionised water Up to 1 Litre - Materials used in the experiment Running Buffer Item Volume Supplier 10% SDS 20 ml Sigma Aldrich Tris/Glycine 10X 200 ml - Deionised water Up to 2 Litres - Materials used in the experiment
compound on the cell migration. A migration assay on BMA 178-2 cells shows that the wound/scratch closed to 90%, 96% and 100% in 24 hours, 48 hours and 72 hours respectively, see Figures 3.25 and 3.26. However, the addition of AM exogenously with three different doses (1µM, 1nM and 1pM) was not able to stimulate or inhibit the migration rate of BMA178-2 cells when compared to the controls, see Figure 3.27. This result is similar to that of the Martínez et al. group, where they found that two different human breast cancer cell lines (T47D and MCF7) transfected with the AM gene did not alter their motility and migration rate, but it was shown to affect the human ovarian cancer cell line (ECV) transfected with the same AM construct (). AM22-52 which is known as the AM antagonist shows no effect on the migration of BMA178-2 in three different doses - 1µM, 1nM and 1pM compared to the control cells, see Figure 3.28. From these results it is plausible to conclude that the concentration of the AM secreted in the media by the BMA178-2 cells was high enough to prevent that action of AM22-52. This contradicts the result of Deng et al. (), where the AM22-52 inhibited the migration of the ovarian cancer cell line HO8910 cells and antagonized the stimulation effect of AM on migration. Conclusion It can be concluded that the BMA178-2 cells express RAMP1, RAMP2, RAMP3 and CLR at the mRNA and protein level which leads to the presence of the AM1, AM2 and CGRP receptors. This result suggests that AM may work in an autocrine manner to stimulate proliferation, migration and invasion as well as to reduce the apoptosis of the BMA178-2 cells through the AM2 receptor because the AM1 receptor, as illustrated in Chapter 1, plays an important role
receptors, resulting in the effect that we have seen in the cancer cells in vitro and in vivo. Further investigation is required to understand the signalling pathways which might be altered as a result of knocking down RAMP3. The results shown here are not without precedent. One group in particular has focused on the use of anti-AM strategies for cancer treatment. Ouafik’s group in France has performed several similar studies, although in some papers the details are sketchy so a full interpretation of the results is difficult. The group used subcutaneous xenograft models for their studies and their treatment groups either received systemic doses of antibodies to CLR, RAMP2 or RAMP3 or a mixture of those. The data on dosage and effects is not clear from the articles, and some animals died, possibly as a result of the effects of a physiological AM blockade (either by a blockade of CLR or AM1 receptors). However, the results indicate that a RAMP3 polyclonal antibody is effective in reducing tumour volume, though less effective than a more comprehensive AM blockade via CLR and AM1R. Further studies show that the injection of these agents into the tumours leads to reductions in tumour volume growth (, ). Finally, a Japanese group has shown that the AM truncated peptide antagonist AM22-52 reduces tumour volume if injected into the tumour mass. The pancreatic cancer cells PCI-43 were injected in SCID mice. 8 days after the cancer cells were injected subcutaneously into the right flanks of the mice, AM22-52 was injected directly into the tumour (intratumoral injection). The injection of AM22-52 continued to day 17. The result showed that tumour formation was inhibited significantly compared to the control (PBS). Also, the diameter of the blood vessels of the formed tumours was reduced hugely compared to control group (). This data illustrates that AM is important for the in vivo tumour formation of pancreatic cancer cells.