Relationship Between Neuropilins Expression And High Hematogenous Metastasis Incidence In Salivary Adenoid Cystic Carcinoma, And Inhibitory Effect Of Propranolol On SACC

BackgroundSalivary adenoid cystic carcinoma(SACC)is one of the most common malignant tumors of the salivary gland.SACC has two prominent clinical characteristics: one is perineural invasion,and the other is high hematogenous metastasis incidence with low lymphatic metastasis incidence.The high hematogenous metastasis incidence of SACC is an important cause of tumor recurrence and poor prognosis,and is also the focus of SACC studies.Intratumoral angiogenesis plays an important role in tumor development and hematogenous metastasis.Besides transporting nutrients,the intratumoral blood vessels provide the channels for tumor cells to metastasis.Intratumoral angiogenesis and micro vascular density(MVD)are associated positively with the incidence of tumor hematogenous metastasis,respectively.Neuropilin family is a class of transmembrane cell receptors,which closely related to tumor angiogenesis / lymphangiogenesis.They participate in tumor cells proliferation,migration,invasion and other biological behavior.NRP1 or NRP2 were involved in the regulation of angiogenesis or lymphangiogenesis in tumors.However,in SACC,it is absence that the researches of NRPs expressions.It is unknown whether there is a difference in the expression of NRP1 and NRP2 resulting in differences in of angiogenesis and lymphangiogenesis in SACC.Beginning in 2008,inhibitory effect on angiogenesis of Propranolol has been found.Now,Propranolol is used as a first-line drug for infant hemangiomas.There are some researches focused on the effect of propranolol on tumor cells.They suggested that propranolol could inhibit tumor cells proliferation,and could promote tumor cells apoptosis.In summary,present study focused on the expression differences among NRP1,NRP2 and their ligands,including VEGF-A,Sema-3A and Sema-3F in normal salivary gland(NSG),oral squamous cell carcinoma(OSCC),non-metastatic SACC and metastatic SACC.We studied the relationship between the proteins expressions and MVD,as well as the relationships among the proteins expressions,MVD and SACC hematogenous metastasis.Meanwhile,the effects of propranolol on SACC cell lines were researched.Objects1.To investigate the expressions of the five proteins,including NRP1,NRP2,VEGF-A,Sema-3A and Sema-3F,in NSG,OSCC,non-metastatic SACC and metastatic SACC;the MVD and LVD in different tissues;the correlation among the proteins expressions,MVD and SACC hematogenous metastasis.2.To investigate the influence of propranolol on the NRP1,VEGF-A and MMP-9 expressions in SACC-83 and SACC-LM;the influence of propranolol on the migration and invasion of SACC-83 and SACC-LM.3.To investigate the influence of propranolol on the proliferation,apoptosis,cell cycle and autophagy of SACC-83 and SACC-LM.Methods1.Using immunohistochemistry staining to study expressions of NRP1,NRP2,VEGF-A,Sema-3A and Sema-3F in NSG,OSCC,non-metastatic SACC and metastatic SACC(30 cases,respectively).Immunohistochemistry staining was also used to study the MVD and LVD in different groups.The differences between the IOD(mean)of NRP1,NRP2,Sema-3A,Sema-3F or VEGF-A and clinical parameters were contrasted using one-way analysis of variance(one-way ANOVA)after data fulfilled equal variance,otherwise,Dunnett's T3 test was used.One-way analysis of variance(one-way ANOVA)was also performed to analyze the differences of MVD and LVD in different groups.The relationship between MVD and the IOD(mean)of the four proteins,including NRP1,VEGF-A,Sema-3A and Sema-3F,were analyzed with Pearson correlation test,respectively.All p values were two-tailed and p < 0.05 was set to be statistically significant.2.Using immunohistochemistry staining to study the expression of ?-AR on tissue sections from SACC(30 cases)and normal salivary gland(30 cases).Western blot assay was used to study the influence of propranolol on the NRP1,VEGF-A and MMP-9 expressions in SACC-83 and SACC-LM.Scratch-wound assays and Transwell chamber cell invasion assay were used to study the influence of propranolol on the migration and invasion of SACC-83 and SACC-LM.3.Using MTT assay to study the influence of propranolol on the proliferation of SACC-83 and SACC-LM;the influence of propranolol on the apoptosis,cell cycle and analyzed by flow cytometry;western blot assay was used to investigate the influence of propranolol on the autophagy of SACC-83 and SACC-LM;the effect of propranolol on the growth of SACC was examined in xenograft tumor in nude mice.Results1.A progressive increase and significant expression of NRP1 was observed in normal salivary gland,non-metastatic SACC and metastatic SACC tissues,as well as the expression of VEGF-A.No significant difference of NRP2 expression was identified among NSG,non-metastatic SACC and metastatic SACC tissues.Sema-3A and Sema-3F were both strongly expressed in the normal salivary gland tissue.The MVD of metastatic SACC tissues was 1.7-fold and 6.2-fold more than that in non-metastatic SACC tissues and NSG(55.5±9.1.vs.32.9±8.0 vs.8.8±3.0,respectively)(p<0.0001).However,there was no significant difference about the LVD among NSG,non-metastatic SACC and metastatic SACC tissues(3.8±1.5 vs.3.9±1.8 vs.4.0±1.1,respectively)(p=0.999)A significant positive correlation was observed between MVD and the IOD of NRP1,as well as the relationship between MVD and the IOD(mean)of VEGF-A.In contrast,there was a significant inverse correlation between MVD and the IOD(mean)of Sema-3s,including Sema-3A and Sema-3F.2.?-ARs were found in SACC and normal salivary gland,respectively.Western blot proved that propranolol can prevent the expressions of NRP1,VEGF-A and MMP-9 in SACC-83 and SACC-LM.Scratch-wound assays and Transwell chamber cell invasion assay proved that propranolol can impair migration and invasion of tumor cells.3.Propranolol can promote the apoptosis and autophagy of SACC-83 and SACC-LM;but,the proliferation was inhibited.In vivo,the mean weight of SACC xenograft tumors,which were treated by propranolol,was significantly lighter than the mean weight of SACC xenograft tumors in control group's(p=0.0029).ConclusionIn SACC,the high level expressions of NRP1 and VEGF-A promoted intratumoral angiogenesis.It provided the necessary conditions for SACC hematogenous metastasis.The low level expression of NRP2 inhibited lymphangiogenesis,blocked the channel of lymphatic metastasis,and reduced the possibility of lymph node metastasis.All of these was the one of causes of SACC has high hematogenous metastasis incidence with low lymphatic metastasis incidence.Propranolol can prevent the expressions of NRP1 and VEGF-A in SACC cells,and prevent intratumoral angiogenesis.On the other hand,the proliferation,migration and invasion of tumor cell can be impaired by propranolol,and the apoptosis and autophagy of tumor cells also be promoted by propranolol.In summary,propranolol can suppress SACC development and hematogenous metastasis through the above ways.