| Hepatocellular carcinoma (HCC) is one of the most prevalent cancers over the world. Most patients could not receive radical therapy because of the late diagnosis. Antiangiogenic therapy, especially sorafenib, became the first-line therapy of advanced HCC. However, sorafenib could only prolong patients’ median survival time in less than3months, in spite of its survival benefit, according to two phase Ⅲ clinical trails. It was demonstrated in preclinical research that sorafenib inhibited the primary tumor along with promoting the metastasis tumor. Our previous study also found that sorafenib could obviously inhibit the growth of the primary tumor and lung metastasis and improve the overall survival, however, it promoted invasive and metastatic potential of HCC. Aspirin, one of the NSAID, was first known to us as the application in antipyretic, analgesia, treatment of rheumatism and prevention of cardiovascular disease. Recently its effect in prevention and treatment of malignant tumor rised into our vision. Our previous study revealed that combination of aspirin and sorafenib significantly suppressed tumor growth and lung metastasis in HCC model, moreover,intrahepatic metastasis induced by sorafenib disappeared after combined with aspirin. This study was performanced to revealed why the increased invasive and metastatic potential of HCC induced by sorafenib could be suppressed by combined with aspirin.Tumor invasion and metastatic is a complex process. Firstly the tumor cell itself must have the strong ability of invasion and metastasis. Secondly the intratumor and paratumor microenvironment stimulated the tumor cell to aggressively invade and metastasis. Lastly the host including the interaction between the platelets and the tumor cells also plays an important role in the process. Therefore, these three fields consist of tumor cell, intratumor and paratumor microenvironment, and the interaction between the platelets and the tumor cells were focused to investigate the mechanism why the increased invasive and metastatic potential of HCC induced by sorafenib could be suppressed by combined with aspirin.1. The increased invasive and metastatic potential of HCC induced by sorafenib was suppressed by aspirin through up-regulating the expression of TIP30in tumor cell.TIP30is a tumor suppressor gene. Over-expression of TIP30in HCC cell line could inhibit the proliferation and metastasis of the tumor cell. It was demonstrated in our previous study that the invasive and metastatic potential was promoted by down-regulating the expression of TIP30in tumor cell which induced its EMT phenotype. In this study, six tumor cell line including LM3, LM3-Lv-Non, LM3-Lv-shTIP30, HepG2, HepG2-Lv-Non and HepG2-Lv-TIP30were used for further investigation. It was revealed that aspirin reversed the EMT phenotype of tumor cell and suppressed the invasive and metastatic potential of HCC which was induced by sorafenib through up-regulating the expression of TIP30. The protein expression of TIP30in the tumor tissue in human HCC nude mouse model was detected by western-blot and Immunohistochemistry, it was confirmed that aspirin up-regulated the expression of TIP30and reduced the intrahepatic metastasis significantly.2. The increased invasive and metastatic potential of HCC induced by sorafenib was suppressed by aspirin through down-regulating the expression of SDF1-α in intratumor and paratumor microenvironment.SDF1, also called CXCL12, is a chemotactic factor. SDF1-α could improve the tumor growth by bonding to its receptor CXCR4. The tumor and paratumor liver specimen of LM3nude mouse model which were treated by sorafenib with or without aspirin were collected, and RT-PCR was performed to detected the mouse derived cytokines related to inflammation. We found that the mRNA level of SDF1-a which was significantly increased in sorafenib group was reduced by sorafenib treatment combined with aspirin. The protein level of SDF1-α in the paratumor liver tissue which detected by WB and the plasma concentration of mouse derived SDF1- a which screened by ELISA assay were in accordance with its mRNA level. Because in vitro assay the expression of SDF1-α in tumor cell was not increased by sorafenib treatment, it was revealed that the elevated amount of SDF1-was derived from the stromal cells. We also found that the tumor cell invasive ability was increased by rhSDF1-α when it was added to the culture solution of the tumor cell. The expression of VEGF in tumor cell was increased by rhSDF1-α as well. In vivo assay, the intrahepatic metastasis increased by sorafenib was obviously suppressed by the CXCR4inhibitor AMD3100. Therefore, the increased invasive and metastatic potential of HCC induced by sorafenib was suppressed by aspirin not only through the influence of the tumor cell itself but also through down-regulating the expression of SDF1-α in intratumor and paratumor microenvironment.3. The increased invasive and metastatic potential of HCC induced by sorafenib was suppressed by aspirin through inhibiting the interaction between the platelets and the tumor cells.The platelets play an important role in the process of tumor invasion and metastatic. Our study investigated that the integrity of tumor vessel was damaged by sorafenib, and the activated platelets overflowed into the tumor microenvironment. In vitro assay, it was revealed that the tumor cell EMT phenotype was induced by the interaction between the platelets and the tumor cells, which lead to the increased invasive and metastatic potential of tumor cell. The adhesive ability between the tumor cells and the endothelial cells was also increased. Combination of aspirin and sorafenib significantly decreased the invasive and metastatic potential of HCC through suppressing the infiltration of platelets in the tumor microenvironment.ConclusionsThe mechanism why the increased invasive and metastatic potential of HCC induced by sorafenib was suppressed by aspirin was explained in the following three aspects: 1. The increased invasive and metastatic potential of HCC induced by sorafenib was suppressed by aspirin through up-regulating the expression of TIP30in tumor cell.2. The increased invasive and metastatic potential of HCC induced by sorafenib was suppressed by aspirin through down-regulating the expression of SDF1-α in intratumor and paratumor microenvironment.3. The increased invasive and metastatic potential of HCC induced by sorafenib was suppressed by aspirin through inhibiting the interaction between the platelets and the tumor cells.The potential application of this workThe mechanism why the increased invasive and metastatic potential of HCC induced by sorafenib was suppressed by aspirin was explained to provide the theoretical and experimental evidence for the clinical application of combination of aspirin and sorafenib in HCC to improve the effect of sorafenib.Originalities of this workIt is the first time to reveal that the mechanism why the increased invasive and metastatic potential of HCC induced by sorafenib was suppressed by aspirin in three aspects including up-regulating the expression of TIP30in tumor cell, down-regulating the expression of SDF1-α in intratumor and paratumor microenvironment, and inhibiting the interaction between the platelets and the tumor cells.
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