The Effect And Mechanism Of Slit-Robo Signal On Oral Squamous Cell Carcinoma And Mucoepidermoid Carcinoma

Slit is a new family of secreted repellents in axon guidance and neuronal migration. Its function is through the Roundabout (Robo) receptor as a chemorepellent in axon guidance and neuronal migration, and as an inhibitor in leukocyte chemotaxis. Slit-2 has been shown to be expression in a large number of solid tumors and Robol expression in vascular endothelial cells. These findings demonstrate the angiogenic function of Slit-Robo signaling and communication between tumor cells and endothelial cells, which could provide a new molecular target for inhibiting tumor's growth. The communication between tumor cells and tumor cells is also known to be important in tumor.The recent study has shown that Slit-Robo signal can regulate the tumor cells, especially in the effect of proliferation and apoptosis of tumor cells. This research is based on the previous research findings that Slit-Robo signal reveals a new mechanism in mediating the crosstalk between tumor cells and endothelial cells, and its function of regulator in the mechanism of proliferation and apoptosis.Our research work is continue to elucidate if the effect of Slit-Robo signal is also be a important role in the carcinogenesis of human oral mucosa. The research work is intended to demonstrate the angiogenic function of Slit-Robo signaling plays a crucial role in the carcinogenesis and discuss the mechanism of the carcinogenesis. We select another oral cancercell Mc3 cell as media to prove the effect of Slit-Robo signal is widely mediating the crosstalk between tumor cells and tumor cells by themselves. The effects of Slit-Robo signal on the proliferation and apoptosis of human mucoepidermoid carcinoma Mc3 cell is pivot and we intend to explore the new mechanism again.Based on these questions, we examined the human samples of different stage as media .The oral carcinoma is one of the most common malignant neoplasma. The normal oral mucosa turns into the carcinoma should be go through the three stages of hyperplasia, dysplasia and carcinoma in situation. We detected the samples of different stage by immunohistochemical staining with the anti-Slit2 antibody, the anti-Robol antibody and the anti-VEGF antibody, and detected the MVD with the expression of the vWF (von Willbrand factor).We want to investigate the expression of the Slit2 and the relationship between the Slit2 and angiogenesis. We select Mc3 cell as media. The human mucoepidermoid carcinoma is one of the most common oral malignant neoplasma in head and neck region .The Mc3 cell came from MEC-1 and its proportion of S phase is increased .It keeps the character of mucoepidermoid carcinoma and the more ability of proliferation and metastasis. In our present research we can observe the effect of the proliferation and apoptosis of tumor cells after the addition of the monoclonal antibodies (mAb) R5 of against Robol receptor extracellular domain.The main research findings of this study are categorized as follows:.. The study on the expression of Slit2,Robol, VEGF and vWF of human samples. 1 > We detected the samples of different stage by immunohistochemical staining with the anti-Slit2 antibody, the result showed the Slit2 expression can be found in the all stages of the carcinogenesis and the expression is increased gradually .The expression of Slit2 protein of the dysplasia is significantly different from the normal mucosa, when the cancer cell breakthrough the basement membrane the expression of Slit2 is highest at both the positive cell number and positive intensity. These results showed the Slit-Robo signal could be started at the stage of the dysplasia and increased gradually with the progress of the carcinoma .The signal expressed obviously at the stage of the CIS(carcinoma in situation )and the highest positive intensity as if the waterfall emerged in the carcinoma. It all showed the Slit-Robo signal play a significant role in the carcinogenesis.2, We detected the samples of different stage by immunohistochemical staining with the anti-Robol antibody, the result showed the Robol expression is weakly positive and has no regulation .There is no significantly different in the five groups. The expression of Robol proteinof the carcinoma is more than other groups, the expression is located at the oral cancer cell , theendothelial cell and the leukocyte.3> We detected the samples of different stage by immunohistochemical staining with theanti-VEGF antibody, the results indicated the expression of VEGF is also increased graduallywith the progress of the carcinoma, the trend is consistent with the expression of the Slit2 .Theresults showed that Slit2 and VEGF have a positive correlation in the carcinogenesis. Theirexpression both increased along with the progress of the carcinoma, and the expression get to thehighest at the stage of the carcinoma. From the stage of the dysplasia there was significantlydifferent from the normal mucosa .The results showed the Slit2 protein may switch from aninhibitory to an angiogenic phenotype, which must attract new vessels in order to accelerate theprogress of the carcinogenesis .Slit may plays a positive role in the angiogenesis of OSCC.4* We detected the samples of different stage by immunohistochemical staining with the anti-vWF antibody. Microvessel density (MVD) was determined in the area of most intensevascularization (hot spot) of each tumor. Our research work testified the number of the MVDincreases along with the degree of malignant in the tumor, which changed obviously at the stageof the CIS and get to the maximum at the carcinoma. The results show that MVD has a positivecorrelation with Slit2 and VEGF in angiogenesis of OSCC, it is potently angiogenic as a result ofthe increased growing of tumor. The results also indicated there was a significant relationshipbetween Slit protein and the angiogenesis of carcinoma.— .. The function of Slit-Robo signal on proliferation and apoptosis of human mucoepidermoidcarcinoma Mc3 cells.1 .. Immunohistochemical analysis showed that SIit2 and Robol proteins were also expressed inMc3 cells . Slit2 is a kind of secreting protein. This conclusion proved again the Slit-Robo signalcan mediate the crosstalk between tumor cells and tumor cells in the human mucoepidermoidcarcinoma.2> The effect of Slit-Robo signal on the proliferation were investigated with cell counting ,MTTassay and colonogenic assay, and the expression of PCNA was measured with flow cytometry.The results showed that the growth and proliferation rate of Mc3 cell decreased in vitro after theaddition of R5 mAb. and the expression of PCNA decreased too. So Slit-Robo signal couldregulate the proliferation of tumor cells to inhibit the cancer growth.3^ After the treatment of the (mAb) R5, the apoptosis of Mc3 cells analysis were measured with flow cytometry , FITC-Annexin V/PI ,Hoechst/PI and DNA fragmentation assay. The expression of caspase-3 protein was observed by flow cytometry. The results showed the apoptotic rates increased and the expressions of caspase-3 protein increased relatively in Mc3 cells which were treated with R5. The cell number in the S phase increased, it indicated the cancer cells were blocked in the S phase. The DNA Ladder and the photo of the Hoechst/PI assay also proved the effect of Slit-Robo signal on the apoptosis. We use the immunofluorescent staining of caspase-3 for flow cytometric analysis, and the expression of the group treated with R5 increased. So Slit-Robo signal plays a crucial role in the development of tumor by apoptosis and caspase-3 protein may be one of the important transductions in apoptosis of Mc3 cells.In summary, the experimental results revealed that the positive expression of Slit2 protein and the degree of malignant have a positive correlation in the carcinogenesis, and the MVD increased along with the expression of the Slit2 and VEGF protein. Slit2 protein may be have synergistic effect on the angiogenic activities, and may play a significant role in the pathogenesis of the oral cancer. We also demonstrated the Slit-Robo signal can widely mediate the crosstalk between tumor cells themselves to regulate the ability of proliferation and apoptosis. These results could lead to the finding of a new way for clinical therapy of human carcinoma.