Based On The Molecular Mechanism Of SCCHN And Metastasis Of Prognostic Effects Of LM609 Mediated Integrin?v?3-CXCR4/CXCR7 Biological Axis

As reported by AJCC, squamous cell carcinoma in head and neck(SCCHN) has been the sixth common malignant tumor. There are 40-50 million new cases annually,most of which are elderly man, and 10-15 million patients died annually. The treatments of SCCHN in different stages are not the same, but they could be divided into surgery, chemotherapy and other treatment. SCCHN in early stage usually has a relatively good prognosis, and requires surgery or precise radiotherapy, there is no difference in local control rate between them.SCCHN in middle stage requires surgery combined with radiotherapy and chemotherapy. Preoperative induction chemotherapy is widely debated, but the latest study compares surgery alone group and the preoperative application of TPF induction chemotherapy group, the recurrence rate and survival rate did not differ significantly, but induction chemotherapy has advantages for improving patients' quality of life. Treatment of advanced cancer is dominated by palliative care, in which the patient usually has been carried out operation or radiotherapy and chemotherapy and other comprehensive treatment, kappa scores were generally low, these patients cannot tolerate more stimulated chemotherapy. Drug targeted therapy, biological therapy or Chinese medicine treatment usually be prescribed to alleviate the patient's illness. Extensive local invasion and distant metastasis are the main failure for cancer treatment.Application of single drug or treatment is not ideal; an important reason is that there is wide signal transduction pathway.Because of high mortality induced by local invasion and distant metastasis in advanced SCCHN, more and more researches focus on how to inhibit the invasion and metastasis of tumor cells. The latest study finds that before tumor cells flow in the body fluids and tissues, it is necessary to overcome the barrier of vascular endothelial cells, including at least four steps(cells with fluid; cell stable adhesion to vascular endothelial; cells through gap between endothelial cells and cell migration to specific tissues), during these processes, cell adhesion and infiltrating are controlled by chemokines selectively, then leading cells to the targeted place, and finally leading to the implementation of promoting wound healing and remove the source of infection, eliminate the abnormal proliferation of cells and maintain the function of tissue cells balance. Similar to the role of chemokines, integrin mainly locates at the cell and extracellular matrix, cell adhesion provides the possibility of cell invasion and metastasis. Metastasis of primary tumor cells is mainly due to a decrease in adhesion between tumor cells; these cells adhere to the basement membrane by integrin, then invade the circulatory system; furthermore, by integrin cells specifically again adhere to selected organs through vein endothelial cells. The cell adhesion ability is constantly change, the main purpose is to promote tumor metastasis,increased or reduced cell adhesion ability can be directly or indirectly regulate tumor cell metastasis, therefore both chemokines and integrinsplay a very important role in cell adhesion.Chemokines and integrin, which belong to the role of transmembrane proteins,have attracted more and more attention to malignant tumor in recent years. Despite more and more attentions have been payed into integrin and chemotaxis, most researches just focus on their mechanism and molecular biological characteristics and neglect their relationship. Recent research confirmed that the integrin family can be prompted by some chemotactic factor receptor, then affecting cell signal transduction by changing the invasion, metastasis, adhesion, migration and other functions. We aimed to explore the role of ?v?3 integrin in the signal way mediated by SDF-1-CXCR4/CXCR7 in SCCHN. We hope to prove whether SDF-1 can activatealpha v beta 3 integrin-CXCR4/CXCR7 signal pathway by application of SDF-1,alpha v beta 3 integrin specific inhibitor of LM609 and CXCR4 inhibitor amd3100,CXCR7 inhibitor CCX754, and to testify the reversal effect of LM609/AMD3100/CCX754 in SCCHN. On the one hand, collecting samples of SCCHN primary tumor,lymph node metastasis, and normal oral mucosa and then evaluating the expression of?v?3integrin/CXCR4/CXCR7 in these tissues, and assessing the UN-QOL and PSS-HN quality of life in patients with high expression, preliminary studying the correlation among the three factors. and finally through a series of in vitro experiments, to find if the expression of alpha v beta 3integrin can be activated by SDF-1 in SCCHN cell line PCI-13; on the other hand, using specific inhibitor LM609/AMD3100/CCX754 on the antagonism of PCI-13 metastasis of SCCHN cell line, we aimed to target to develop new drugs for head and neck squamous cell carcinoma, to provide a theoretical basis.Method1. Using immunohistochemical SABC to analysis the expression of?V?3integrin?CXCR4?CXCR7 in primary head and neck squamous carcinoma,lymph node metastases, normal oral mucosa tissues and normal lymph nodes, and collecting the general statistical data of the patients.2. Using methyl azo wow salt light absorption method(MTT) to detect the effect of SDF-L and M609/AMD3100/CCX754 on the proliferation of SCCHN PCI-13 cell.3. Using flow cytometry method to detect the effect of SDF-1 and LM609 /AMD3100 / CCX754 on the expression of integrin ?v?3 of SCCHN PCI-13 cell.4. Using Western-Blot test to determine the effect of SDF-1 and LM609 /AMD3100 / CCX754 on the protein expression of SCCHN PCI-13 cell.5. Using artificial tissue wound healing assay and Transwell chamber method to determine the effect of SDF-1 and LM609 / AMD3100 / CCX754 on the migration of SCCHN PCI-13 cell.6. Using Transwell chamber assay plus Matrigel to determine the effect of SDF-1 and LM609 / AMD3100 / CCX754 on the invasive ability of SCCHN PCI-13 cell.7. Using Inverted fluorescence microscope to observe the role of SDF-1 and LM609 / AMD3100 / CCX754 on the ability of changing cytoskeleton of SCCHN PCI-13 cell after FITC / Phalloidin / PI staining.8. Using PSS-HN and UW-QOL scale to assess the quality of life and survival rate of patients with postoperative defect caused by CXCR4 / CXCR7 / integrin ?v?3expression in head and neck squamous cell carcinoma, and the patients of low / no expression of CXCR4 / CXCR7 / integrin ?v?3 as the control group.Result1. integrin?v?3 / CXCR4 / CXCR7 staining mainly located in SCCHN cell cytoplasm and cell membrane, and the strength of expression was statistically associated with tumor size, lymph node metastasis, surrounding tissue invasion, and it was not statistically relevant with age, gender and other general information.2. MTT assay showed after induction by SDF-1, the proliferation rate of PCI-13 of SCCHN cell line was significantly higher, but after the inhibition of LM609/ AMD3100 / CCX754, the proliferation was significantly decreased, of which the rate of inhibition was different, and the inhibition rate of AMD3100 was similar to CCX754 and LM609 was the highest.3. Flow cytometry showed integrin ?v?3 and CXCR4 / CXCR7 were highly expressed in PCI-13, and the expression was on the cell membrane and cytoplasm.And flow cytometry and Western-Blot experiments confirmed that the expression of integrin ?v?3 in PCI-13, after the SDF-1, induced a significant increase, but after LM609 / AMD3100 / CCX754 suppression, integrin ?v?3 expression of PCI-13 levels were significantly decreased.4. Wound healing assay and Transwell chamber experiments found that after SDF-1-induced stimulation, the migration of PCI-13 cells was significantly increased,compared with the control group, the scratch width 28 H / 48 H were significantly reduced. However after LM609 / AMD3100 / CCX754 inhibition, scratch width of PCI-13 significantly decreased than SDF-1 induced group, but the rate of inhibition were different, and the inhibition rate of AMD3100 was similar to CCX754 and LM609 was the highest. The result of cell invasion assay in Transwell chamber wassimilar to the above ones.5. Under the inverted microscope after FITC / Phalloidin / PI staining, the number of PCI-13 cells wire / lamellipodia significantly increased after the SDF-1induced. But after LM609 / AMD3100 / CCX754 suppression, wire / lamellipodia sufficient number decreased significantly. Further experiments demonstrated that inhibition after LM609 plus SDF-1-induced, and then after AMD3100 / CCX754 inhibition experiments found that the wire / lamellipodia number of inhibitory effect and did not change significantly, but after AMD3100 / CCX754 suppression plus SDF- 1 after induction, and then after LM609 inhibited its silk / lamellipodia number of inhibition rate changes, further reduced. From this level of positive and negative findings prompt, CXCR4 and CXCR7 may play it induces laminin cytoskeletal changes by the action of ?v?3 integrin.6. UW-QOL was measured and found that the preoperative score in the control group is generally higher than the experimental group, but after 1 month and 6months after surgery its rates were low, in part because of the higher expectations in the control group. After 12 months in the control group, the most satisfied results were the appearance, response and anxiety, while the experimental group is the appearance of pain and anxiety, while the experimental group is the appearance, pain and anxiety, of which the reason is that the pain disappeared after excision,preoperative informed and comprehensive disease removed.7. PSS-HN measured preoperative study, which showed that 77.2 divided into an experimental group of normal diet, 70.4 and 49.4 for the experimental group voice and social scenarios eating, essentially flat with the control group, but normal eating and eating at social occasions of the experimental group were significantly lower 12 months after operation. As the postoperative recovery time, there were higher and higher scores, higher and higher degree of satisfaction, but the speech function has the biggest change. With the high rate of lymph node metastasis, 3 year survival rate was slightly lower.Conclusion1. The expression of integrin?v?3 / CXCR4 / CXCR7 in head and necksquamous cell carcinoma primary tumor and lymph node metastases showed positive correlation, providing a theoretical basis and laid a certain experimental basis for further exploration of the mechanisms of integrin?v?3 / CXCR4 / CXCR7 involved in lymph node metastasis of SCCHN.2. CXCR4-integrin?v?3 signaling axis and CXCR7-integrin?v?3 signaling axis may be induced by SDF-1, prompting increased expression of integrin?v?3. LM609 inhibited the effect of SDF-1 on the expression increased role to integrin?v?3.3. CXCR4-integrin?v?3 and CXCR7-integrin?v?3 signaling axis may be induced by SDF-1, prompting the increase of proliferation, chemotaxis, invasion which is the key step to the head and neck squamous cell carcinoma lymph node metastasis, and this effect can be inhibited by LM609.4. The inhibition of LM609 and AMD3100 / CCX754 was different, after the replacement of the inhibition order; LM609 inhibition performance was more prominent, suggesting that integrin?v?3 was likely downstream signaling of CXCR4/ CXCR7-integrin?v?3 signaling axis.5. The reconstruction quality of life assessment score of the patients of high expression of integrin?v?3 / CXCR4 / CXCR7, using PSS-HN and UW-QOL scale,was significantly higher than the patients of moderate-low expression of integrin?v?3/ CXCR4 / CXCR7. With the high rate of lymph node metastasis, 3 year survival rate was slightly lower.6. PSS-HN and UW-QOL scale score of the patients after flap surgery showed that the highest satisfaction scores of the appearance and the lowest satisfaction scores of the verbal, followed by eating swallowing function, to a certain extent that the success in shape, but on the specific use of the function needing to be improved.7. The level of expression of integrin ?v?3 / CXCR4 / CXCR7 was closely related to the level of quality of life scores, helping us to assess the extent of surgery preoperative and postoperative follow-up observation time, directly or indirectly affecting care and prognosis of patient.