| Background & ObjectiveStudies have indicated that the decreased platelets inhibited tumor growth and metastasis through clinical observation and animal experiment more than half a century ago,however the approach could not be employed because thrombocytopenia can cause hemorrhage. P-selectin, which express on stimulated endothelial cells and activate platelets, plays an important role on platelets binding to tumor cells and tumor cells adhering to vascular endothelial cells. Can P-selectin inhibit tumor growth and metastasis? And it is unclear whether P-selectin inhibits tumor growth through antagonizing P-selectin-mediated platelets adhesion in tumor or not. Serum amyloid P component (SAP) and C-reactive protein (CRP) are the acute-phase proteins secreted by hepatocytes in response to inflammatory reaction. SAP and CRP, the two plasma proteins of pentraxin family, are synthesized principally in hepatocytes and up-regulated by a repertoire of cytokines, such as interleukin-1 and 6 (IL-1 and 6) and tumor necrosis factor-α(TNF-α), in inflammatory status. Geng laboratory previously showed that P-selectin precipitates a 28-kDa glycoprotein from the membrane lysate of human platelets and identified this molecule as SAP. SAP specially binded to P-selectin, and inhibited P-selectin-mediated cell adhesion and inflammatory reaction. Then, can interaction of SAP and P-selectin inhibit tumor growth through antagonizing P-selectin-mediated platelets adhesion? The current study aimes to reveal the effect of P-selectin on tumor growth and metastasis, specifically the effect of P-selectin and SAP on tumor growth through antagonizing P-selectin- mediated platelets adhesion. Methods1 The effect of P-selectin on tumor growthThe B16 cells were passaged in MEM media with 10% FCS. Cells (1×105) were detached with 5 mM EDTA/PBS, disaggregated into single-cell suspensions, and injected s.c. into the lower flanks of 6- to 8-week-old mice, which were kept in a pathogen-free environment with antibiotics in their water. Tumors were measured along the plane of the body cavity and tumor volume was calculated as: [(length)×(width)2]/2. The mice were tracked for up to 15 days, when the largest tumor masses exceeded 20 cm3, and euthanasia was required by institutional guidelines.Female P-selectin-/- mice in a C57 background were mated with male Rip1-Tag2+/- mice in a C57 background. F1s were crossed and their offspring were genotyped for Tag+/- and P-selectin+/- null mutants. Tag+/- and P-selectin+/- null mutants were crossed to P-selctin-/- mice in C57 background and their offspring were genotyped for Tag+/- and P-selectin-/- null mutants,which were PKO/Rip1-Tag2 mice. Enumerate of angiogenic islets and determinate of tumor burden of PKO/ Rip1- Tag2 mice and Rip1-Tag2 mice.2 The effect of P-selectin on tumor metastasisB16 cells were detached with 5 mM EDTA and disaggregated in PBS. Persistent cell aggregates settled down within minutes, and single cells remaining in the supernatant (1×105) were injected into the tail vein. After 3 weeks, the mice were sacrificed, and their brains, lungs, livers, kidneys, and enlarged dorsal lumps were collected for analysis. Half of the tissue sample was analyzed histologically for metastasis.3 The effect of platelet on insulinomas of Rip1-Tag2 miceRip1-Tag2 mice were injected using antiplatelet antibody-GPIbalpha antibody, which led to thrombocytopenia of experiment mice. Then enumerate of angiogenic islets and determinate tumor burden of experiment and control mice.4 The distribution of platelet in pancreas of PKO/ Rip1-Tag2 and Rip1-Tag2 miceIsolate the pancreas of PKO/ Rip1-Tag2 and Rip1-Tag2 mice. After frozen section, we detected the expression of platelet marker CD41 with immunofluorescence. 5 The effect of P-selectin-mediated platelets adhesion on tumor growthIn order to study P-selectin-mediated platelets adhesion on tumoe growth, we injected the platelets of P-selectin knockout mice and C57 mice into PKO/Rip1-Tag2 mice. Then we enumerated of angiogenic islets and determinated of tumor burden of PKO/ Rip1-Tag2 mice.6 The effect of SAP on tumor growth through antagonizing P-selectin-mediated platelets adhesion in tumorFemale SAP +/+ mice in a C57 background were mated with male Rip1-Tag2 +/- mice in a C57 background and progeny carrying the Rip1-Tag2 transgene and the mutant SAP gene were enumerated of angiogenic islets and determinated of tumor burden.Results1. Initially the studies in P-selectin knockout mice showed that a palpable tumor derived from the injected 1×105 s.c. B16 cells within 1 week and grew slowly in size thereafter. We next studied the groups of 6-8-week-old P-selectin-/- mice. Tumor growth velocity was reduced significantly in the absence of P-selectin. In the P-selectin-deficient group did death incidence rate reduce while significantly improved survival rate . And P-selectin-/- reduced tumor burden compared with the 6-,8-,12- and 14-week Rip1-Tag2 mice. The PKO/Rip1-Tag2 mice had a significant decline in tumor burden compared with the Rip1-Tag2 mice. And overall survival of PKO/Rip1-Tag2 mice was increased.2. The metastasis foci number of Liver and lung in P-selectin knockout mice was fewer than that of transplantation tumor of C57 mice. 3. After injecting antiplatelet antibody-GPIbalpha antibody and control antibody into Rip1-Tag2 mice, we found tumor volume of Rip1-Tag2 mice injected GPIbalpha antibody was significantly decreased compared with Rip1-Tag2 mice injected control antibody.4. There were more platelets aggregated in insulinoma of Rip1-Tag2 mice than that in insulinoma of PKO/Rip1-Tag2 mice.5. The tumor number and volume of PKO/Rip1-Tag2 mice, which was injected PKO platelets, were fewer than that of PKO/Rip1-Tag2 mice injected C57 platelets.6. The angiogenic islet and tumor number of Rip1-Tag2 mice was more than that of SAP-Tg/Rip1-Tag2 mice. And the tumor vloume of of Rip1-Tag2 mice was bigger than that of SAP-Tg/Rip1-Tag2 mice.Conclusions1. P-selectin deficiency attenuated the tumor growth.2. P-selectin deficiency attenuated the tumor metastasis.3. Platelets promoted the growth of Rip1-Tag2 tumor.4. P-selectin promoted the growth of Rip1-Tag2 tumor through P-selectin-mediated platelets adhesion in tumor.5. SAP inhibited tumor growth through antagonizing P-selectin-mediated platelets adhesion in tumor.
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