| Primary lung cancer, commonly known as lung cancer, is the most common cause ofcancer-related death in the world, and its incidence and mortality rates are still on the riseeach year. Poor prognosis of patients with lung cancer is mainly due to the distanthematogenous metastasis. However, the mechanism of the distant hematogenous metastasis isstill unclear in lung cancer. Therefore, in order to find the potential thearapy for the treatmentof lung cancer, we need to further the study of the mechanism of the distant hematogenousmetastasis of lung cancer.Hematogenous metastasis is a complex multi-step and multi-factor process, involvinglocal invasion, intravasation of cancer cells into blood vessels, circulation, recognition of afavorable distant microenvironment, extravasation, invasion of the extracellular matrix,dormancy, and subsequent secondary growth at a distant site. In recent years, many researchessuggested that the platelet activation in the circulation was associated with the hematogenousmetastasis in various malignancies, such as lung cancer. Thus the platelet activation might bea new therapeutic target. However, how platelet activation is involved in the thehematogenous metastasis of tumor cells is still unknown.When activated, platelets gathered around the tumor cells, and form the tumor cellcomplexes by adhering to tumor cells with a variety of biological active substances andadhesion molecules also involved. P-selectin is a cell adhesion molecule (CAM) on thesurfaces of platelets and endothelial cells, and mainly exists in α-granules in unactivatedplatelets with no or low expression at rest. When platelets are activated, P-selectin can rapidlyexpress on the membrane surface of platelets by membrane fusion. Thus, P-selectin is abiomarker for the platelet activation. Recent studies demonstated that P-selectin couldinfluence the activity of tumor metastasis by binding to the surface ligands of tumor cells,including colon cancer, breast cancer, malignant melanoma, gastric cancer and lymphoma.However, most surface P-selectin ligands of tumor cells haven’t been completely identifieduntil now. And it is still unclear about how the interactions between P-selectin and its ligand affect the hematogenous metastasis of lung cancer. Therefore, it is worth further study.At first, we investigated the expressions of P-selectin on activated platelets in patientswith lung cancer, and explored the relationship between the expressions and theclinical-pathological features, hematogenous metastasis and prognosis. Secondly, theexpression of P-selectin glycoprotein ligand-1(PSGL-1) in lung cancer tissues and lungcancer cells were detected, then after pSilenser–PSGL-1–shRNA vector was construct andused to transfect into A549cells, we investigated the effect of the P-selectin and PSGL-1interaction on the forming of the tumor cell complexes, on the surface rolling and adhesion ofA549cells, and on the transmembrane invasion of the lung cancer cells. Finally, we furtheranalyzed the interactions between P-selectin and PSGL-1, and the expression change of thehematogenous metastasis-related integrin gene (α3, α5and β1) and the matrixmetalloproteinase genes (MMP-2, MMP-9). Through the above steps, we aimed to identifythe role of P-selectin expression on activated platelets in the hematogenous metastasis of lungcancer, to preliminary explore the mechanism involved in the effect of the P-selectin bindingto PSGL-1on hematogenous metastasis of lung cancer. Our research may contribute to thefurther study of the mechanism underlying the hematogenous metastasis of malignant tumorcells, and provide an experimental basis for finding a new therapeutic target of lung cancer.Methods:1) To explore the correlation of the P-selectin expression on activated platelets to theclinical-pathological features and hematogenous metastasis of lung cancer.a) We analyzed the correlation between the periphery blood platelet count (BPC) and theclinical-pathological features in the lung cancer patients, who turn first to the hospital.b) We extracted and purified, and investigated the expression of the P-selectin positivepercengtage on activated platelets (PPP) to the lung cancer patients by flowcytometric analysis, and bivariate correlations and logistic regression mothods wereused respectively to analysis the risk of PPP to the distant hematogenous metastasis.c) We investigated the effect of different factors on the survival rate of patients by thefollow-up analysis through the Cox proportional hazard model and Kaplan-Meiermethod.2) To explore the effect of the interaction between the activated platelet P-selectin and its ligand PSGL-1on the adhesion and invasion activity of the lung cancer cells.a) The expression of PSGL-1was determined by Laser scanning confocal microscopy orwestern-blot analysis in the different type tissues and lung cancer cells.b) The pSilenser–PSGL-1–shRNA vector was construct by shRNA technology, and usedto transfect into A549cells to build the PSGL-1shRNA transfected A549cells.c) After platelets and A549cells were co-cultured in vitro, we observed the forming ofthe tumor cell complexes under scanning electron microscope, and investigated theeffect of the P-selectin and PSGL-1expression on the forming of the platelet-tumorcell complexes, on the surface rolling and adhesion of A549cells on vascularendothelial cells by the parallel plate flow analysis, and transwell assays were used toobserve the transmembrane invasion and dispersal ability of the lung cancer cells.3) To explore the effect of the interaction between activated platelet P-selectin and PSGL-1on the hematogenous metastasis-related genes in lung cancer.a) After platelets with different intervention factors and A549cells were co-cultured invitro, we investigated the effect of the interaction between P-selectin and PSGL-1onthe mRNA and protein expressions of integrin α3, α5and β1by RT-PCR andwestern-blot, and analyzed its clinical meaning.b) After platelets with different intervention factors and A549cells were co-cultured invitro, we also investigated the effect of the interaction between P-selectin and PSGL-1on the mRNA and protein expressions of MMP-2and MMP-9by RT-PCR andwestern-blot, and analyzed its clinical meaning.Results:1) The periphery BPC and the P-selectin expression on activated platelets in patients withlung cancer significantly increased, especially in the lung adenocarcinoma groupcompared with the control group (p <0.01).2) There was a significant correlation between the P-selectin expression and thehematogenous metastasis of lung cancer (p <0.05). And the periphery BPC and theactivated platelet P-selectin expression were the independent influence factor related tohematogenous metastasis. The lung cancer patients with high P-selectin expression (≥44%) had significantly a higher risk of hematogenous metastasis than those with low P-selectin expression (<44%) under multiple analysis (OR=3.696,95%CI,1.39-9.83, p<0.01).3) The survival time was significantly shortened in patients with high P-selectin expressioncompared with those with low P-selectin expression (p<0.001). The significant differencewas also observed in the survival curve between high and low P-selectin expressionpatients. High P-selectin expression was the independent high-risk prognostic factor inpatients with lung cancer under the Cox proportional hazard model (Hazard Ratio=5.98,95%CI,2.12-16.87, p=0.001).4) The P-selectin ligand PSGL-1was detected in both lung cancer tissues and in-vitro lungcancer cells. The strongest expression of PSGL-1was found in the lung adenocarcinomasamples, next in the squamous cell carcinoma samples, and less in small cell lung cancersamples.5) After co-culture of the activated platelets and A549cells, the forming of platelet-tumorcell complexes was observed, and significantly different between the high and lowP-selectin expression cells. The forming of platelet-tumor cell complexes wassignificantly inhibited by anti-P-selectin antibody or PSGL-1shRNA method.6) The activated platelets promoted the capture and adhesion of A549cells on the surface ofthe umbilical vein endothelial cells (HUVEC). The promoting effect was positivelycorrelated with the P-selectin expression on activated platelets. Compared with thecontrol group, the cell rolling speed reduced as much as60.9%(p <0.05), and the celladhesion number increased as much as245.9%(p <0.01). The cell rolling and adhesionability of lung cancer cells on the HUVECs decreased after the application ofanti-P-selectin antibody or PSGL-1shRNA method.7) After co-culture of the activated platelets and A549cells, the transmembrane invasionability of lung adenocarcinoma cells changed. After the transmembrane interactions, thecell invasion number significantly increased36.9%in A549cells with high P-selectinexpression on activated platelets (p <0.01), while the adhesion and invasion activity ofthe lung cancer cells decreased in those A549cells with P-selectin expression inhibitedby anti-P-selectin antibody or PSGL-1shRNA method.8) After co-culture of the activated platelets and A549cells, high P-selectin expression onactivated platelets significantly promoted the mRNA and protein expression of integrin α3 and α5, especially integrin α5. The promoting effect was inhibited in those A549cellswith P-selectin expression inhibited by anti-P-selectin antibody or PSGL-1shRNAmethod.9) After co-culture of the activated platelets and A549cells, high P-selectin expression onactivated platelets also significantly promoted the mRNA and protein expression ofMMPs, especially MMP-9. When the interaction between activated platelet P-selectinand PSGL-1was blocked, the expression of MMP-9also significantly decreased.Conclusions1) The periphery blood platelet count and the activated platelet P-selectin expression werethe independent influence factor related to the hematogenous metastasis of lung cancer,especially lung adenocarcinoma. High P-selectin expression on activiated platelets wasthe independent high-risk prognostic factor in patients with lung cancer.2) High platelet P-selectin expression could induce the forming of platelet-tumor cellcomplexes, enhance the capture and adhesion of lung cancer cells on the surface of thevascular endothelial cells, increase the transmembrane invasion ability of lung cancercells, and thus ulteriorly affect the potency of hematogenous metastasis of lung cancer.3) The interaction between activated platelet P-selectin and PSGL-1might be involved inthe mechanism about how the platelet activation affects the hematogenous metastasis oflung cancer. Both anti-P-selectin antibody and PSGL-1shRNA method could inhibit theforming of platelet-tumor cell complexes, the capture of lung cancer cells on the surfaceof endothelial cells, and the transmembrane invasion ability of lung cancer cells.4） The interaction between P-selectin and PSGL-1might enhance the adhesion of lungadenocarcinoma cells on the surface of the vascular endothelial cells by increasing theexpression of integrin α3, α5, and promote the lung adenocarcinoma cells across theextracellular matrix and basilar membrane by increasing the expression of MMP-9, whichmay be one of the main mechanisms of activated platelet P-selectin promote distantmetastasis of lung cancer. |
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