| Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer in the worldwide and the third in China. According to the prediction of WTO, prevalence of HCC will maintained sustained growth and the second leading cause of death[1] Despite the advancement of cancer therapy, such as surgery, intervene therapy, liver transplantation (LT), the five-year survival rate of patients with HCC is still stayed under 15%[2.3] More than 60% HCC patients were in advanced stage as they are diagnosed, thus losing operation opportunity. Consequently, researches of anticancer drug are of paramount importance and play the key role in further improving the survival rate.Tumorigenesis, development, invasion and metastasis of tumors are complex and dynamic processes. Chemotherapy is paid more and more attention to for its advantages and the demand for the drugs with good efficacy of anti-cancer metastasis and less side effects is increasing. A growing amout of researchers set their sights on nature products. These natural products are one of the important sources of potential cancer chemotherapeutic and chemopreventive agents with 70% of current anticancer drugs originate from natural products[4]. In addition, drug combination therapy that has the advantage of increasing efficiency of antitumor, delaying drug resisitence and less drug toxicity supply new strategies for antitumor.In the study, we evaluated the efficacy of polymeric nanoparticle encapsulated formulation of curcumin (NanoCurcTM, NC) alone and in combination with sorafenib in biological behaviors of hepatocellular carcinoma. CCK8, wound-healing, modified Boyden chamber, were examined to determine the effect of NC and/or sorafenib in the proliferation, invasion and migration of HCC. Cell cycle and apoptosis were detected by flow cytometric assays. The in vivo effects of the co-treatment in tumor growth and lung metastasis rate were examined using orthotopic and subcutaneous xenograft liver tumor model. At last, we discussed the probable mechanisms for the anti-tumor effects of NC and/or sorafenib, providing new target for HCC prevention and treatment.Part I The impact on HCC biological characteristics by NC combined with sorafenibIn order to explore the impact of NC and/or sorafenib on tumor growth, migration, invasion, cell cycle and apoptosis, we used Huh7and MHCCLM3 cell lines in vitro experiments.After treatment with NC and/or sorafenib, CCK8, wound-healing and modified Boyden chamber was used to determin the capacity of polieration, migration and invasion respectively. Cell cycle and apoptosis were further detected by flow cytometric assays. NC and/or sorafenib could significantly increased the percentages of the early apoptotic, late apoptotic and necrotic of cells, and the percentages of apoptosis cells of the combination group were significantly higher than NC or sorafenib alone. Compare with single agent, the proportion of G2/M phase in combination group was significantly higher (P<0.01).Above results show that NC in combination with sorafenib synergistically inhibited HCC cells proliferation, migration and invasion and increased the percentages of the early apoptotic, late apoptotic and necrotic of cells. The effect of inhibition of HCC cells was related with G2/M arrest.Part II The impact on nude mouse xenograft model biological characteristics by NC combined with sorafenibIn order to explore the impact of NC and/or sorafenib on tumor growth and metastasis in vivo, we used orthotopic and subcutaneous xenograft liver tumor model.Explore the effect of NC and/or sorafenib on tumor growth by establishing orthotopic and subcutaneous xenograft liver tumor model. In the orthotopical and subcutaneous HCC model using MHCCLM3 cells transfected with red. Red fluorescent protein (RFP) gene, we detected the metastatic lesions of HCC in the lung tissues under fluorescence microscopy and validated the results by H&E.The results showed that the volume of orthotopic tumors of the treated mice were much smaller than those of the controls (NC vs sorafenib vs combination vs control: (0.75±0.14) cm3 vs (0.50±0.11) cm3 vs (0.45±0.09) cm3 vs (1.26±0.13) cm3); The number of the lung metastasis rate were 50.0%,66.7%,16.7% and 100% respectively.These indicated that NC in combination with sorafenib can significantly inhibited tumor growth and metastasis in vivo.Part III The underlying mechanisms of NC combined with sorafenib impact on HCCIn order to understand how NC combination with sorafenib inhibited the proliferation, invasion and migration of HCC, we discussed the underlying mechanism in two aspects from pathways and liver cancer stem cell (LCSC). On one hand, NC and sorafenib synergistically enhanced anticancer activity through inhibiting ERK1/2 phosphorylation effectively by reducing NF-kB DNA-binding activities, which led to MMP-9 downregulation. On the other hand, it also significantly decreased the population of CD 133+ cells.We first researched ERK1/2/NF-kB/MMP9 signaling pathways of Huh7 and MHCCLM3 cell lines by RT-PCR, ELISA and Western blot and further validated it with MMP-9 inhibitor (TIMP-1). Secondly, we detected these targets in nude mice HCC tissue by RT-PCR, ELISA and Western blot and immunohistochemical staining. Lastly, flow cytometric assays and immunohistochemical staining were used to detect the population of CD 133+ HCC cells in cell lines and HCC tissue.These results showed that NC and sorafenib synergistically enhanced anticancer activity through inhibiting ERK1/2 phosphorylation effectively by reducing NF-kB DNA-binding activities and led to MMP-9 downregulation in vivo and vitro, which indicated that NC in combination with sorafenib might synergistically regulate tumor proliferation and invasion through this signaling pathway. TIMP-1 expression was most in the combination group. In addition, NC in combination with sorafenib inhibited CD133~+ HCC cells in not only cell lines but also HCC tissue.In summary, NC in combination with sorafenib inhibited cell invasion and metastasis synergistically through ERK1/2/NF-kB/MMP-9 signaling pathway and decreased the population of CD133+ HCC cells, which prevented recurrence. |
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