| Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in Asia and Africa. Despite endeavors have been made to improve its prognosis, the overall survival, however, is still unsatisfied. The high rate of metastasis and recurrence after curative resection was responsible for the poor prognosis of resectable HCC. Anti-angiogenesis is an attractive treatment for HCC, because HCC is a typical hypervascular cancer, its growth, metastasis or even hepatocarcinogenesis depend on angiogenesis. Angiogenesis inhibitors have been hailed as opening a new era in cancer therapy. But a flurry of animal studies suggests that such drugs may in certain situations actually accelerate the spread of cancer.The phase III sorafenib SHARP trial as well as oriental sorafenib trial finally demonstrated a significant survival benefit, making sorafenib the new reference standard for systemic therapy of patients with advanced HCC. However, the survival benefit and a retardation of tumor progression were only about three months. As an angiogenesis inhibitor, it should be clarified whether sorafenib promote metastasis.Using different orthotopic human hepatocelluar carcinoma nude mice model with deferent metastatic potential, we investigated the mechanism of tumor progression under sorafenib treatment, and the effect of sorafenib on metastatic potential of HCC. As tumor metastasis can be also affected by tumor microenvironment, including the inflammatory cytokines or macrophages. Therefore, we investigated both tumor cell and tumor associated macrophages, and to explore clinical available approach to improve the effect of sorafenib in HCC.1. Low-dose sorafenib promote local invasion and metastatic potential of HCC.The purpose of this part is to investigated different dosage of sorafenib on invasion and metastatic potential of sorafenib with human HCC orthotopic nude mouse model with different metastatic potential. In a patient-like model LCI-D20, we found low-dose sorafenib and high-dose sorafenib significantly inhibited tumor growth and prolonged survival of mice bearing tumors. Sorafenib reduced number of metastatic lesion in lung but did not decrease lung metastatic rate. Because sorafenib-treated tumor was much smaller than that of control, when applying standardized lung metastatic rate (number of lung metastasis/tumor volume) we found sorafenib significantly promoted metastasis potential to lung. Low-dose sorafenib also induced intrahepatic metastasis, but this phenomenon was not observed in high-dose sorafenib group.To confirm this effect, we reconducted sorafenib treatment using red-or green-fluoresce protein infected tumor cells established in our institute LM3-RFP (high metastatic potential) and HepG2-GFP (low metastatic potential) and found that low-dose sorafenib did significantly promoted intrahepatic metastasis with no reduction of abdomen metastatic rate or lung metastatic rate. Low-dose sorafenib also induced significant increased circulating tumor cells as measured by flow cytometry.2. Hypoxia induced EMT is one mechanism of sorafenib to promote invasion and metastatic potential of HCC.Immunohistochemistry staining for HIF-la and Pimodazole revealed significant hypoxia induced by sorafenib. Hypoxia promotes metastasis by inducing EMT. In LM3-RFP and HepG2 model, sorafenib induce significant change in expression of a panel of EMT markers, including decrease of E-cadherin and elevation of N-cadherin and Vimentin, indicating enhanced invasion and metastatic potential of HCC undergoing sorafenib treatment.3. Down regulation of HTATIP2 and evasion of apoptosis induced by sorafenib as important molecular basis.Tumor Pathway Finder PCR Array was applied to screen possible key genes responsible for sorafenib induced invasion and metastatic potential. Twelve genes was significantly changed by sorafenib, and 6 of them were antioncogene HTATIP2,SERPINB5,SYK,Rb1,FAS,P53 and 1 was pro-apoptosis gene TNF. Since most of the antioncogenes were related to P53 and function as pro-apoptosis genes, we postulated that antioncogene down-regulation may help tumor cells evading apoptosis. 4. Aspirin up-regulated HTATIP2 to revert the metastatic promotion effect of sorafenib.Aspirin has been revealed to induce apoptosis by up-regulating P53 and is clinical available, so we combined aspirin with sorafenib. Combination of aspirin and sorafenib significantly suppressed tumor growth and reduced lung metastasis in HCC model, and intrahepatic metastasis induced by sorafenib disappeared after combined with aspirin. RT-PCR was applied to evaluate gene level change of antioncogene and found aspirin only up-regulated HTATIP2-mRNA, but P53 and other antioncogenes were unaffected by aspirin. Down-regulation of HTATIP2 was also confirmed by western blotting at protein level. So we postulated that HTATIP2 was responsible for anti-metastatic potential effect of aspirin when combined with sorafenib.We constructed LM3 cells infected with LV-sh HTATIP2, LV-shNon, and started treatment with sorafenib or combination of sorafenib and aspirin after orthotopically inoculation. We found LM3-LV-shHTATIP2 model had more invasive tumor compared with LM3-wt tumor, and sorafenib did not induce more intrahepatic metastasis compared to control. And after treat by combination of aspirin, intrahepatic metastasis was reduced to a less extent compared with that in the LM3-wt model (50% vs.100). Aspirin also reduced lung metastasis in combination with sorafenib, but to a less extent compared with that in the LM3-wt model.5. In vitro experiments demonstrated apoptosis induction effect of combination of aspirin and sorafenib.We treated LM3 cells with sorafenib 5μM by a time course of 0h,3h,6h,9h and 12h, and revealed a reduction of HTATIP2 was most prominent in 3-6h. LM3-LV-shHTATIP2, LM3-LV-shNon and LM3-wt cell lines were exposes to 5μM sorafenib or 0.1 mM/0.5 mM aspirin, HTATIP2-mRNA was measured by RT-PCR and apoptosis were evaluated by Annexin V-PI using flow cytometry. Aspirin reverted down-regulation of HTATIP2 by sorafenib, enhanced apoptosis and reduced invasion cells when combined with sorafenib compared to sorafenib alone. 6. Macrophage depletion enhanced effect of sorafenib by antiangiogenic and antimetastatic effect.Although sorafenib significantly inhibited tumor growth, it induced elevation of F4/80-and CD11b-positive cells in peripheral blood and increased infiltration of intratumoral macrophages, which was associated with elevation of plasma VEGF, SDF-la and CSF-1, suggesting that macrophages played a role in the progression of tumor undergoing sorafenib treatment. With depletion of macrophages by clodrolip or zoledronic acid, tumor progression, tumor angiogenesis, and metastasis were significantly inhibited compared with that of mice treated by sorafenib alone. Zoledronic acid is clinical available and is promising to be combined with sorafenib for HCC patients.Conclusions:1. Low-dose sorafenib promoted invasion and metastatic potential in human HCC nude mouse models with different metastatic potential.2. HTATIP2 down-regulation contributed to enhanced metastatic potential of HCC induced by sorafenib.3. Combination of aspirin with sorafenib promoted apoptosis and reverted sorafenib's pro-invasion and pro-metastasis effect by down-regulating HTATIP2.4. Zoledronic acid enhanced antitumor effect of sorafenib by macrophage depletion.The novelties of this work:1. Sorafenib promoted metastatic potential by down-regulating HTATIP2 in two orthotopic human HCC nude mouse models.2. Combination of aspirin enhanced antitumor effect of sorafenib by promoting apoptosis and inhibiting invasion and metastasis through up-regulating HTATIP2.3. Macrophage recruitment and infiltration contributed to evasive resistance in mice undergoing sorafenib treatment. Macrophage depletion enhanced antitumor effect of sorafenib. |
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