| Background and objectives:Nasophryngeal carcinoma (NPC) is one of the common malignant tumors in China, especially in the southern part of the country. Radiotherapy is considered to be a prevalent treatment in present. In recent years, with advances in computer technology and the upgrade of the accelerator equipment、 through the three-dimensional conformal radiation therapy (3-dimensional conformal radiotherapy,3-DCRT) technology and toneconformal radiation therapy (intensity modulated radiotherapy, IMRT) are come true. The3-DCRT technology is characterized by distributing the radiation dose in the three dimensions of the space consistented with the tumor shape. But the IMRT is a kind of new technology which is complying distribution consistented with tumor shape while the dose intensity distribution can also be adjusted. For this, the survival rate of nasopharyngeal carcinoma was significantly improved, especially the five-year survival rate of patients with early staying up to80%-90%. However, some clinical patients present local recurrence and local residue after radiotherapy, one of the important reasons is due to the radiation resistance in cancer cells. Autophagy, or "self-eating," is a very ancient phenomena in the course of life evolution, which existed widely in yeast and other lower creatures, is an important way in which protein degradated and organelles eliminated to keep cell homeostatic, it is also a process to get rid of cancer cells in mammals. It was first described as an important cellular process almost50years ago in Greece. Datas from recent years indicated that autophagy is considered to be another Programmed cell death path to distinguish from cell apoptosis, represents a dynamic lysosomal pathway responsible for degrading organelles and long-lived proteins. Autophagy is a very primitive biological phenomena in the normal cells physiological processes. Many micromolecule protein participated in autophagy, what we understood well is Beclinl gene and Beclinl protein, microtubule-associated protein light chain3(MAPLC3) and p62is another mark for autophagy some immunodetection..The basal autophagy level of cells keep lowly to maintain homeostatic situationin usually, autophagy level is up-regulated rapidly when crisis come. For example, Nutritional deficiencies^growth factor deficiency、 cell reconstruction or too much damaged cell organelle or metabolic waste.The main regulater of nutrition sensor regulates autophagy is:target of rapamycin(TOR)kinase and eukaryotic initiation factor2(eIF2)kinase Gcn2. TOR play an important role as a gate keeper in cell growth and inhibite autophagy occur. TOR shut off autophagy signal when nutrition adequacy. While eIF2start autophagy signal when starvation.There are many genes lies down-stream of TOR kinase,which participate autophagy some development、 confluence、 maturement、recycling. Rapamycin and the analogue is antagon for TOR receptor and induced autophagy, while Chloroquine disphosphate (CDP) and3-methyladenine (3MA) were usually used to inhibit autophagy formation. Autophagy level is closely related cancer cells occurrence, development and sensitivity of cancer therepy. At early stage of cancer cell formation, provoked autophagy help toeliminate damaged cell organelle^and degrade the noxious substance in endochylema (eg:damaged mitochondria) as well, pretect normal cells from cancerization. However, in late stage of tumor development, autophagy offers precursors such as amino acids, fatty acids and nucleotides to be recycled and used for macromolecule synthesis. It can also block the mitochondrial apoptosis signaling cascade spread, eventually lead to the tumor cells to evade apoptosis. It can be activated as adaptive response to adverse environmental conditions, such as chemotherapy and radiotherapy, down-regulation of autophagy level would make organelles elimination breakdown and accelerate cell death to improve therapeutic efficacy.In order to investigate the relationship between autophagy and NPC and the possibility sensitization effect of radiotherapy after autophagy expression alteration, the cellular and molecular biology methods were used to detected the autophagy-related protein expression level of CNE-2cells to elucidate the relationship of autophagy level and irradiation; investigated changes of radiotherapy sensitivity and autophagy correlated protein when autophagy level of CNE-2altered. We also detect the CNE-2cells apoptosis rate to explain its mechanism to explain the association between autophagy and apoptosis. To investigate the protective function of autophagy in the CNE-2cells apoptosis induced by irradiation.Methods1. Firstly, western-blot were used to detect the expression level of autophagy-related protein LC3-Ⅱ(microtubule-associated protein1light chain3,MAP1-LC3-Ⅱ) and p62in nasopharyngeal carcinoma CNE-2cells, which were under different concentration Chloroquine disphosphate(CDP) and different concentration rapamycin treated. Then, Flow cytometry (FCM) was used to detect the ration of apoptosis of CNE-2cells, which were treated with three different conditions.2. Western-blot and Flow cytometry (FCM) were used again to detect autophagy and apoptosis in nasopharyngeal carcinoma CNE-2cells which were under different doses (0,2,4,6,8and lOGy) X-ray radiation at different time (24h and48h) to determine the relationship between cell autophagy and apoptosis after irradiation. Then, Western-blot and FCM were used again to detect the level of autophagy-related protein LC3-Ⅱ and p62expression and the ration of apoptosis of CNE-2cells, which were treated with six different conditions as irradiation only(10Gy), Chloroquine disphosphate(CDP) treated only, rapamycin treated only, Chloroquine disphosphate combine with lOGy irradiation, rapamycin combine with10Gy irradiation and the blank control group. Which were in order to define the protection function of autophagy in CNE-2cells.Results1.when CNE-2cells was treated with40P M Chloroquine disphosphate (CDP), the expression level of LC3-Ⅱ and p62protein both increased more pronounced than other CDP groups (P<0.01). In rapamycin treated groups, the level of LC3-II protein expression was significantly increased after20nM rapamycin treated, but the level of p62expression decreased, there were significant differences between various groups (P<0.01). And the ration of apoptosis is no difference among control group,40y M CDP group and20nM rapamycin group (P>0.01).2. With the increase of irradiation doses (0,2,4,6,8and lOGy) and the elongation of irradiation time (24and48h), the phenomenon of autophagy increased significantly. While the apoptosis rate of CNE-2cells were increased with irradiation dose at48h post-irradiation, there were significant differences between various groups(P<0.05), Compared with irradiation alone, Chloroquine disphosphate(CDP) combined with irradiation significantly increased cell apoptosis, as well as p62and LC3-II protein level. However, rapamycin combined with irradiation induced strong upregulation of LC3-II expression but decreased of cell apoptosis and p62level. The ration of cells apoptosis among only(10Gy) irradiation, Chloroquine disphosphate(CDP) treated only and rapamycin treated only groups is no significant differences (t=-1.198; t=-3.222; t=-1.902, p>0.05).Conclusion Firstly, Chloroquine disphosphate can inhibit autophagy in CNE-2cells? the inhibiton is conspicuous at40μM concentration. While rapamycin can induce autophagy and the induction is extraordinary at20nM concentration. Otherwise, at the two conditions, it will not impact the ration of apoptosis of CNE-2cells. Secendly, irradiation can induce autophagy and apotosis in CNE-2cells. Autophagy level is increased depend on the radiation dose and exposure time. When the single dose is less then or eaqual6Gy, the cells apoptosis ration is also increased belong dose and time. Third, inhibition of autophagy increased irradiation-induced apoptosis, while induction of autophagy protected cell survial. It suggests that autophagy might play a role as a self-defense mechanism in cancer radiotherapy. |
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