The Role And Mechanism Of Autophagy Inhibition Combination Antiangiogenesis On Hepatocellular Carcinoma Therapy

Human primary hepatocellular carcinoma (HCC) is one of the highly prevalent malignant diseases worldwide, particularly in our country, which carries a very poor prognosis and high recurrence. Surgery is the first choice for HCC, but it was always too late for the patients when they were identified with HCC. Chemotherapy and radiotherapy are two of the three major means for treatment of primary liver cancer, especially for those patients who is not suited to surgery. But the result is far from satisfaction, as hepatocellular carcinoma is usually resistant to chemotherapeutics and radiotherapy. It is significant to find a new anticancer target and method to improve HCC therapy and patients'survival. Recently,Over the past several years, there has been an explosion in our understanding of the molecular alterations occurring in HCC and, coupled with the availability of new'targeted'agents, there is great promise that patient outcomes will be improved. Several HCC studies have correlated elevated VEGF levels with the biological behavior of the disease and clinical outcomes. Elevated VEGF levels have been detected in the serum and cancerous tissue of patients with HCC. These data suggest a role for dysregulated angiogenesis in HCC and point to the potential role of disrupting VEGF signaling in the management of HCC,and new drugs were designed. But the biologic effects of anti-angiogenic agents in HCC were limited when used as a single-agent.As a protective mechanism, autophagy plays an active role to keep cells alive under nutriment deprivation, ischemia, hypoxia and some other pathological conditions. It's possible that Beva treatment induced ischemia could up regulate autophagy in HCC, which in turn plays a protect role in the therapy. Accord to this hypothesis, this study proceeded from the following three aspects: 1. Examine the effect of Beva on HCC xenografts'growth and autophagy genesis; 2. The role of autophagy in nutriment deprivation-tolerance of hepatocellular carcinoma cells; 3. The Role and Mechanism of Autophagy Inhibition Combination Antiangiogenesis (Beva) on Hepatocellular Carcinoma TherapyPart 1 Examine the effect of Beva on HCC xenografts'growth and autophagy genesis First of all, we established SMMC7721 xenografts model on nude mice. We treatment the mice with Bevacizumab (Beva), as a result, we found Beva indeed has therapy effect on SMMC7721 xenografts. We next examined the inter effect of Beva on tumors with immunohistochemisty and western blot methods, the result shows that Beva reduced blood vessels density and induced autophagy in SMMC7721 xenografts.Part 2 The role of autophagy in nutriment deprivation-tolerance of hepatocellular carcinoma cellsThis part of experiments were designed to use the EBSS culture media to analogue the nutriment stress made by Beva. We selected 3-MA as the autophagy inhibitor, and transected plasmid expressing GFP-LC3 which can be used as a marker of autophagy into hepatocellular cells SMMC-7721 and Hep3B. We observe the position of GFP-LC3 on cell membrane under fluorescence microscope to examine autophagy indirectly, to confirm the efficiency of autophagy inhibition of 3-MA, and the EBSS culture media's effect on autophagy induction in hepatocellular cells. We next examined the cell viability of hepatocellular cells cultured with EBSS and 3-MA by CCK-8, the result indicated that nutrient stress combination autophagy inhibiton reduced the cell viability of hepatocellular cells compared with nutrient stress alone.DAPI staining and PI-AnnexinV staining with flow cytometry were took out to quantization the cell apoptosis, and the resule indicated that hepatocellular cells cultured with EBSS and 3-MA had a increased apoptosis rate compared with EBSS treat alone. We further demonstrated this phenomenon by transfected si-beclin1 into SMMC-7721 and Hep3B cells. The results from light microscope and DAPI staining also confirmed what we found above. Thus we got the conclusion: autophagy plays a protect role in hepatocellular cells during nutrient stress, and inhibition of autophagy could lead to apoptosis of hepatocellular cells treated with nutrient deprivation.Part 3 The Role and Mechanism of Autophagy Inhibition Combination Antiangiogenesis (Beva) on Hepatocellular Carcinoma TherapyChloroquine (CQ) was selected as autophagy inhibitor in vivo, due to it;s stability and efficiency on autophagy inhibition. We established SMMC7721 xenografts model on nude mice, and examined the therapy effect of Beva alone and Beva combination with CQ on SMMC7721 xenografts. The results shows that the combination group has a better therapy effect compared with Beva treatment along, both the weight and volume of tumor were decreased notablely. The further exploration by immunohistochemisty showed that the proliferation ability was remarkably reduced in combination treatment group (using PCNA antibody). TUNEL stanting and immunohistochemisty by actived-caspase3 antibody demonstrated the apoptosis rate was significantly increased in CQ and Beva treatment group compared with Beva treatment along. These results demonstrated that autophagy inhibition could synergism the therapy effect of Beva, and this could be a strategy on anti-angiogenesis therapy of HCC.To sum up, we conclude as follows:1. Bevacizumab (Beva) treatment unregulated autophagy in SMMC7721 xenografts, which could be a protect mechanism on tumor cells.2. Autophagy inhibition could reduce the cell viability and induce the cell apoptosis of hepatocellular cells cultured under neutrino stress in vito, which suggest the combination of anti-angiogenesis agents with autophagy inhibitors for a better therapy in vivo.3. Autophagy inhibitor combination with anti-angiogenesis agents displayed a more efficient result on SMMC7721 xenografts. This result proved a potential strategy on anti-angiogenesis therapy of HCC.