| Human primary hepatocellular carcinoma (HCC) is one of the highly prevalent malignant diseases worldwide which carries a very poor prognosis and high recurrence. Chemotherapy is one of the three major means for treatment of primary liver cancer, for example transcatheter arterial chemoembolization (TACE) has become the first choice of unresectable hepatocellular carcinoma and is one of the most important therapies for that of advanced stage. Although hepatocellular carcinoma is not sensitive to chemotherapy, and its mechanism is unclear now.The occurrence and development of hepatocellular carcinoma and treatments such as TACE is closely related with hypoxia. On one hand, hypoxia occurs when the aberrant blood vessels can't supply the rapid growing tumor mass with adequate amounts of oxygen; on the other hand, treatments such as blood vessels embolization and anti-angiogenesis aggravate this hypoxia. Changes in gene transcription are induced by hypoxia, then a series of responses are switched on, this not only changes the biological characters of tumor but also increase the resistance of tumor to radio- and chemotherapy. Recently, autophagy is reported to perform a vital role in protecting cells against adverse conditions, including hypoxia.As an protective mechanism, autophagy plays an active role to keep cells alive under nutriment deprivation, growth factor deprivation, hypoxia and some pathological conditions. Autophagy is an cellular process that cells degrade impaired organelles and macromolecules with lysosomes, providing energy, amino acids and macromolecular precursors for cells to maintain the balance of protein metabolism and cellular homoeostasis. During autophagy, the formation of autophagosome is critical. The formation and elongation of autophagosome involves two ubiquitin-like conjugation systems: the Atg5-Atg12 complex and the microtubule-associated protein light chain 3. The microtubule-associated protein light chain 3 (LC3), the mammalian ortholog of yeast Atg8, localizes on the surface of autophagic vesicle.Just as the normal ones, cancer cells can also sense the hypoxia, activate the signal pathway and then induce the expression of associated genes to overcome this adverse condition. Hypoxia-inducible factor-1(HIF-1) works as the center in hypoxia signal pathway. Signal pathway of hypoxia and autophagy interact with each other to regulate the expression of intracellular protein, and then promote the survival of cells in adverse condition. Then dose the chemoresistance of hepatocellular carcinoma cells have something with the hypoxic microenvironment in carcinoma? If so, whether the existed and aggravated hypoxia by treatments such as blood vessel embolization and anti-angiogenesis in tumors attributes to poor prognosis? And how the hypoxia mediates the chemoresistance? To solve these problems, this study proceeded from the following three aspects: 1. examine the effect of hypoxia on chemoresistance of hepatocellular carcinoma cells; 2. investigate the role of autophagy in chemoresistance of hepatocellular carcinoma cells in hypoxia in order to answer the question that why the hepatocellular carcinoma cells are non-sensitive to chemotherapy; 3. study the relation between hypoxia, autophagy, apoptosis and chemoresistance, explore the mechanism of chemoresistance of hepatollular carcinoma cells in hypoxic condition in the molecular level.Part 1 Effect of hypoxia on chemo-sensitivity of hepatocellular carcinoma cells First of all, treat hepatocellular carcinoma cells SMMC-7721 with chemotherapeutic agents: cisplatin, epirubicin, mitomycin and gemcitabine of varied concentration for different time, and then observe the effect of these chemotherapeutic agents on cell survival and the relation between the dose, exposed time and cytotoxicity of these chemotherapeutic agents; next, select appropriate concentration and exposed time of the chemotherapeutic agents, according to the result before, to treat hepatocellular carcinoma cells SMMC-7721 and HepG2 in 1% O2, examine the difference of cytotoxicity caused by chemotherapeutic agents in 1% O2 and 20% O2. The result suggests that under some certain concentrations and exposed time, the cell death caused by chemotherapeutic agents in normoxia is much higher than that in hypoxia, exhibiting significant chemoresistance of hepatocellular carcinoma cells in hypoxia. Moreover, this chemoresistance is commonly found in hepatocellular carcinoma cells when treated by various chemotherapeutic agents, not some chemotherapeutic agent specific, strongly identify the chemoresistance of hepatocellular carcinoma cells in hypoxia.Part 2 Role of hypoxia-induced autophagy in chemoresistance of hepatocellular carcinoma cellsStudy in this part aimed to solve the problem that which mechanism mediates the chemoresistance of hepatocellular carcinoma cells in hypoxia. Culture SMMC-7721 cells in 1% O2 and 20% O2 respectively, with the addition of chemotherapeutic agents, then observe the morphologic changes in cell apoptosis by DAPI staining, analyze the cell apoptosis quantitatively by PI-AnnexinV staining with flow cytometry, and analyze the cell cycle by PI staining with flow cytometry, thus we can discuss the relation between cell apoptosis, cell cycle and chemoresistance of hepatocellular carcinoma cells mediated by hypoxia. On the other hand, transfect plasmid expressing GFP-LC3 which can be used as a marker of autophagy into hepatocellular cells SMMC-7721 and HepG2, then incubate under 1% and 20% O2, and treat with cisplatin of 20μM whose chemoresistance is most significant in this study according to the result before. At the end of the treatment, observe the position of GFP-LC3 on cell membrane under fluorescence microscope to examine autophagy indirectly. Finally, inhibit autophagy with autophagy inhibitor 3-MA, and then analyze the occurrence of autophgy and cell apoptosis after the inhibition, investigate the role of autophagy induced by hypoxia in chemoresistance of hepatocellular carcinoma cells. The attained data show that the apoptotic potential of hepacellular carcinoma cells reduced in hypoxic condition, and the cells survival is promoted. The result about cell cycle show that there is not obvious difference between treatment groups in hypoxia and normoxia, suggests the chemoresistance of hepatocellular carcinoma cells in hypoxia provided in our study has nothing to do with cell cycle. Observation under fluorescence microscope showed that hypoxia can induce autophagy obviously, but not normoxia. And the further study with 3-MA showed that the chemoresistance mediated by hypoxia is attenuated significantly, as the cell apoptosis caused by chemotherapeutic agents in normoxia is similar with that in hypoxia when autophagy was inhibited by 3-MA. All of these suggest that it is autophagy as an important mechanism mediates the chemoresistance of hepatocellular carcinoma cells in hypoxia.Part 3 Analysis in molecular level for the effect of autophagy induced by hypoxia on chemoresistance of hepatocellular carcinoma cellsAt first, we examine the expression of HIF-1αin hypoxia and normoxia by Western Blotting, found that 1% O2 could induce the up-regulation of HIF-1αwhile 20% O2 almost can't induce its expression. In order to investigate whether HIF-1 participates in the autophagy of hepatocellular carcinoma cells in hypoxia, we construct a HIF-1αspecific siRNA to transfect SMMC-7721 cells to inhibit the expression of HIF-1 in hypoxia, then examine the expression of autophagy associated gene Atg5 by RT-PCR, as a result, mRNA of Atg5 is down-regulated significantly after the inhibition of HIF-1α, meanwhile observation under fluorescence microscope also showed the decrease of LC3Ⅱ. So HIF-1αis involved in the regulation of autophagy in hypoxia. At last, we examine the expression of the critical molecular in apoptotic pathway-caspase3 in hypoxia and normoxia and when HIF-1αwas inhibited by siRNA or not. The data showed that compared with that in normoxia, expression of caspase3 is much lower in hypoxia when exposed to chemotherapeutic agents, but after inhibition of siRNA/HIF-1α, the expression of caspase3 increased, even in hypoxia, equal to that in normoxia after the treatment of chemotherapeutics. Thus the molecular pathway in chemoresistance of hepatocellular carcinoma cells in hypoxia is clarified primarily, and this make the foundation for further study of molecular mechanism in chemoresistance of hepatocellular carcinoma cells mediated by autophagy in hypoxia, and also provide the experimental evidence for exploration of new methods for treatment of hepatocellular carcinoma in hypoxia.To sum up, we conclude as follows:1. Hypoxia enhances the chemo-resistance of hepatocellular carcinoma cells to a certain extent, this result suggests that the regional hypoxia caused by rapid tumor growth and treatment such as TACE affects the prognosis of chemotherapy.2. Hypoxia can induce the autophagy of hepatocellular carcinoma cells, and just this autophagy confers the chemoresistance of hepatocellular carcinoma cells in hypoxic condition, and this chemoresistance can be reversed while autophagy is inhibited. For the mechanism, autophagy induced by hypoxia promotes the cell survival in adverse condition, and reduced the cells apoptotic potential, which contributes to the resistance to chemotherapeutic agents.3. The process of hypoxia induce autophagy to enhance the chemoresistance of hepatocellular carcinoma cells is executed through the interaction between HIF-1, Atg and caspase. |
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