The Study Of The Radiation Effect Related Microenviroments Of Head And Neck Tumor And The Response Of Adjacent Normal Tissue

BACKGROUND AND OBJECTIVEAs one of the treatments of tumors,radiotherapy plays a very important role in the treatment of head and neck cancer.Although the overall survival of paitents with head and neck cancer has been significantly improved,but recurrence after treatment and distant metastasis is still the major causes of death in head and neck cancer patients.Currently there are two major challenges for cancer radiotherapy including radiation resistance and the limit on dose caused by damage of normal tissue adjacent to tumor cells.Lots of studys show that the microenvironment of tumor cell has an important influence on the response of tumor to chemotherapy or radiotherapy.It has also shown that the majority of solid tumors have a hypoxic area,which is very common in head and neck cancer.As an important feature of the tumor microenvironment,hypoxia gives tumor cells with special biological function,and it is thought to be a critical factor for confer radioresistance to tumor cells.Renin-angiotensin system(RAS)is an important peptide-based system that has been recognized for decades as a principal determinant of arterial blood pressure,and fluid and electrolyte balance.Recent studies have found that the presence of local RAS in various tissues of human,these tissues contain cells that can express renin,angiotensinogen,ACE or proteases,and can generate Ang II through ACE-dependent or independent pathways.Local RAS plays an important role in regulating organization's structure and physiological action.Accumulating data from in vitro,animal and clinical studies has shown that the RAS is frequently dysregulated in malignancy,and this correlates with poor patient outcomes.The presence of Ang II in a number of tumor tissue or mouse xenograft model has been found by researchs.Ang II can induce the expression of vascular endothelial growth factor(VEGF),GLUT1 and HIF-1,which is involved in the development of several chronic diseases.In tumor tissue,these factors usually associated with tumor hypoxia microenvironment.In most solid tumors,depending on the tumor size and the formation of blood vessels,the tumor microenvironment contains normoxic area,hypoxic area(normal glucose or low glucose)and necrosis.Therefore,we hypothesized that Ang II may play an important role in the tumor hypoxia microenvironment.This study demonstreated that the existence of a local RAS in the head and neck tumor hypoxia microenvironment.We also clarified the effect of local RAS on tumor radiosensitivity through regulation of HIF-1? in head and neck cancer cells.Our study provided a new and strong theoretical basis for hypoxia-induced radioresistance in head and neck cancer and introduces a new strategy to overcomer adioresistance in head and neck cancer patients.The dose delivered to tumor tissue was limited due to the appearance of acute and late toxicities caused by radiation,such as mucositis,dysphagia,xerostomia,dermatitis,hearing loss and pulmonary fibrosis,which may have profound effects on the patient's life span and quality of life.So it is very important to clarify the response of normal tissue to radiation.High-precision radiotherapy(HPR),i.e.,intensity-modulated radiation therapy(IMR.T),has steadily established its role in cancer treatment.Increasing clinical data have shown the advantage of HPR due to its excellent dose distribution in the target volume and sparing of normal tissues.Compared to conventional external beam radiotherapy(EBRT),HPR usually requires more fraction delivery time(FDT)due to the more complicated delivery process(approximately 1 to 3 minutes for conventional EBRT and 15 minutes or more for HPR,i.e.,prospective respiratory gating Image-guided IMRT usually takes more than 50 minutes to deliver the same fraction dose).Prolonged FDT has been demonstrated to reduce radiation-induced cell death in cancer cells through increased repair of sublethal lesions.However,it is unclear whether irradiation with prolonged FDTs produces an protective effect on acute-and late-responding normal tissue.Acute-and late-responding normal tissues have different ?/? ratios and usually generate a distinct biological response to different patterns of radiation.Radiation-induced sensorineural hearing loss(SNHL)is a common,late,and permanent complication in cancer patients managed with radiotherapy.In addition,hair cell damage caused by irradiation of the inner cochlea is the major reason for SNHL.Radiation-induced severe skin response and oral mucositis are common acute adverse effects of radiotherapy for cancer;they are thought to result from excessive inflammation and epithelial ablation,including keratinocyte damage caused by radiation therapy.Thus,we used HEI-OC1 hair cells as late-responding tissue cells and HaCaT human keratinocytes as acute-responding tissue cells to investigate the response of different normal tissues to irradiation with prolonged fraction delivery times.Autophagy represents a dynamic lysosomal pathway responsible for degrading organelles and long-lived proteins.In addition,it is important in maintaining intracellular homeostasis and cell health,which can be activated as an adaptive response to adverse environmental conditions,such as deprivation of nutrients,hypoxia and different types of therapeutic stress.During the last decade,the number of studies on the roles of autophagy in human diseases and its cellular mechanism has become extensive.Hou et al.showed that autophagy may play an important role in protecting stemness of mesenchymal stem cells from irradiation injury.But the role of autophagy in radiation induced acute-and late-responding tissue damage is unclear.Our results indicated that late-responding normal tissue cells benefitted more from prolonged FDTs compared with acute-responding tissue cells,which was mainly attributed to enhanced cytoprotective autophagy mediated via the ATM-AMPK signaling pathway.CHAPTER 1 The biological function and molecular mechanism of local RAS on hypoxia-mediated radioresistance of head and neck cancerPURPOSETo study the biological function and molecular mechanism of local RAS on hypoxia-mediated radioresistance of head and neck cancer.METHODS1.Detection of Angiotensin ? in the hypoxia microenvironment of head and neck cancer:Expression of Ang ? was detected by Elisa and immunofluorescence assay in CNE1 and CNE2 cells in different microenvironments(including normoxia,hypoxia with normal glucose or low glucose).Xenografts in nude mice were built with CNE2 cells,and correlation between Ang ? and tumor hypoxia microenvironment was studied by immunofluorescence analysis of Ang?and HIF-1?.2.Relationship between Angiotensin ? and radiosensitivity of head and neck cancer cells:Relationship between Angiotensin II and radiosensitivity of head and neck cancer cells was determined using the colony-forming assay.Increasing numbers of cells were seeded in 6-well culture plates.After irradiation,the cells were incubated in an undisturbed state for 10-14 days.Colonies were fixed and stained with crystal violet,and the number of colonies with more than 50 cells was scored by microscopic inspection.The surviving fraction was calculated according to the multi-target single-hit model using GraphPad Prism 5.0 software.Xenografts in nude mice were built to investigate the effect of candersatan on tumor cell survival with CNE2 cells.3.Detection of the mechnisim involved in the regulation of tumor cell radiosensitivity by Ang ?.The signaling pathways involved in the regulation of tumor cell radiosensitivity by Ang II were investigated using western blot assay.Whole cell protein lysates were extracted using radio-immunoprecipitation assay(RIPA)buffer containing proteinase and phosphatase inhibitor cocktails and quantified using a bicinchoninic acid protein assay kit.Equivalent amounts of total protein were resolved via SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes.Blots were incubated with the indicated antibodies and visualized using an ECL detection kit(Millipore).4.Statistical analysisIn the experimental studies,the data are presented as the means ± standard deviation.The samples were analyzed using Student's t test or analysis of variance(ANOVA).Tumor volumes were analyzed using repeated measurement of variance analysis.All statistical analysis was performed using SPSS 13.0 software,and a P value of<0.05 was considered to be statistically significant.The qualitative data were representative of more than three independent experiments that were performed in triplicate.Results:1.The local RAS was activated in tumor hypoxia microenvironment.The level of Ang ? of CNE1 and CNE2 cells in different oxygen concentrations was detected by ELISA kits.The results showd that there was only a small amount of Ang ? in the culture medium of CNE1 and CNE2 cells in normoxia condition,and the level of Ang ? in the culture medium significantly increased when cells exposed to hypoxia with normal or low glucose.Immunofluorescence assay showd that only a small amount of Ang ? was expressed in the cytoplasm of cells under normoxia conditions,and the level increased significantly in hypoxia cells.Further,fluorescence assay in a mouse xenograft model showd that Ang ? was expressed in the regions where also expressed HIF-1?.These results suggested that the local RAS was activated in tumor hypoxia microenvironment.2.The local RAS regulated the radiosensitivity of head and neck cancer cells.Clonogenic assay showd that compared to cultured in normoxia condition,CNE2 cells were more resistance to radiotherapy when cultured in hypoxia condition with normal or low glucose,and AT1R antagonist candesartan can significantly enhance the radiosensitivity of CNE2 cells in hypoxia condition with normal or low glucose.But candesartan did not significantly increase the radiosensitivity of CNE2 cells under normoxia condition,and Ang ? could significantly decrease the radiosensitivity of CNE2 cells in normoxia condition.AT1R antagonist candesartan combined with radiotherapy also decreased the tumor volume compared to the control group only treated with radiotherapy.These results suggested that the local RAS activated in tumor hypoxia microenvironment may mediate the radioresistance of tumor hypoxia cells.3.The local RAS regulated the HIF-1? in tumor hypoxia microenvironment.Western blot assay showed that Ang ? or exposed to hypoxia can significantly increase the expression of HIF-1? protein in CNE2 cells,and AT1R antagonist candesartan can inhibited the expression of HIF-1? protein under hypoxia condition.Further,we tested the signaling pathways involves in regulating the expression of HIF-la and the results showd that the MAPK and PI3K/Akt signaling pathways,which were reported to have the ability to improved the mRNA translation of HIF-1?,were actived after treatment with Ang ? or exposed to hypoxia condition.The AT1R antagonist candesartan can suppressed the activation of these two signal pathway under hypoxia condition.These results suggested that local RAS regulate the HIF-1?in tumor hypoxia microenvironment through activating the MAPK and PI3K/Akt signaling pathway.Conclusion:This study demonstreated that the activaion of a local RAS in the head and neck tumor hypoxia microenvironment.We also clarified the effect of local RAS on tumor radiosensitivity through regulation of HIF-1? in head and neck cancer cells.CHAPTER2 Impact of prolonged fraction delivery times on radiation-induced toxicity on two normal tissue cell linesPURPOSETo investigate the impact and mechanism of prolonged fraction delivery times(FDTs)on radiation-induced toxicity in normal tissue cell lines.METHODS1.Irradiation:To compare the cell-killing effectiveness of irradiation-simulating conventional EBRT and HPR with different FDTs,fractionated irradiation of 0 Gy,2Gy×1,2Gy×2,2Gy×3,2Gy×4,and 2Gy×5 was given with one fraction per day,similar to the clinical dose-time-fractionation pattern.In irradiation simulating conventional EBRT,the fraction dose of 2 Gy was delivered continuously.In irradiation simulating HPR with different FDTs,each fraction dose of 2 Gy was given in 8 equal subfractions at a dose rate of 2Gy/min and inter-subfraction intervals of 2,5,and 7 minutes,such that the total FDT was 15,36,or 50 minutes.To investigate the effect of different FDTs on cell apoptosis,DNA damage and autophagy,a dose of 10 Gy was given in 8 equal subfractions at a dose rate of 5 Gy/min;the total FDT was 2,16,37,or 51 minutes.2.Colony formation assays:Clonogenic survival of normal tissue cells was determined using the colony-forming assay.Increasing numbers of cells were seeded in 6-well or 96-well culture plates.After irradiation,the cells were incubated in an undisturbed state for 10-14 days.Colonies were fixed and stained with crystal violet,and the number of colonies with more than 50 cells was scored by visual inspection.Dose-survival curves for single-dose irradiations were plotted according to the standard linear-quadratic model using GraphPad Prism 5.0 software(GraphPad Software,San Diego,CA),and those for fractionated irradiations with 2 Gy per fraction were manually plotted.3.Apoptosis Assay:FITC-conjugated Annexin V was used to detect the presence of apoptosis.The cells were seeded in 6-cm dishes one day prior to radiation exposure.After irradiation,the cells were incubated for 24 h and then harvested and stained using the Annexin V-FITC/PI apoptosis detection kit(Invitrogen,Inc.,Carlsbad,CA,USA)according to the manufacturer's instructions.The resulting fluorescence was detected using flow cytometry.4.Western blotting analysis:Whole cell protein lysates were extracted using RIPA buffer containing proteinase and phosphatase inhibitor cocktails and quantified using a bicinchoninic acid protein assay kit.Equivalent amounts of total protein were resolved via SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes.Blots were incubated with the indicated antibodies and visualized using an ECL detection kit(Millipore).Membrane densitometric quantification of the western blot signal intensity was performed using Image Pro Plus 6.0 software.5.DNA damage assay:Cells were cultured on coverslips and subjected to treatments as indicated.Cells were fixed and stained with a primary antibody for anti-?-H2AX(Abcam Inc.,Cambridge,MA,USA)as previously described#.All matched samples were examined using laser scanning fluorescence confocal microscope(Olympus,Japan).?-H2AX foci were quantified and averaged from ten images(×60),which were obtained from three independent experiments.6.GFP-LC3 Assay:Cells were plated into 24 wells and transfected with GFP-LC3 using a lentiviral vector for 24 hours,and cells stably transfected with GFP-LC3 were screened for GFP-LC3 Assay.Cells were irradiated and then fixed in 4%paraformaldehyde.Fixed cells were examined under a laser scanning fluorescence confocal microscope(Olympus,Japan).Ten images(×60)were randomly selected from three independent experiments to determine the average number of GFP-LC3 punctate dots per cell.7.Determination of ROS generation:Changes in intracellular ROS levels were determined by measuring the oxidative conversion of cells permeable to 2',7'-dichlorofluorescein diacetate(DCFH-DA)to fluorescent dichlorofluorescein(DCF).Cells were grown in 96-well culture plates at an optimal density of 5 x 103 cells/ml with 200 ?l of culture medium per well.One day after plating,the cells were exposed to irradiation and then incubated for an additional 24 h.After removing the medium,the cells in the plates were washed with PBS and incubated with DCFH-DA at 37? for 20 min.Cells were washed with PBS and DCF fluorescence was subsequently detected at an excitation wavelength of 488 nm and an emission wavelength of 525 nm using a fluorescent microplate reader(SpectraMax M5,Sunnyvale,CA,USA).8.Statistical analysis:In the experimental studies,the data are presented as the means ± standard deviation.The samples were analyzed using Student's t test or analysis of variance(ANOVA).All statistical analysis was performed using SPSS 13.0 software,and a P value of<0.05 was considered to be statistically significant.The qualitative data were representative of more than three independent experiments that were performed in triplicate.Results:1.Acute-and late-responding normal tissue cells exhibit differential sensitivity to prolonged fraction delivery times.The clonogenic surviving fraction was measured in HEI-OC1 cells and HaCaT cells,and the results showd that the surviving fractions after irradiation simulating HPR with a prolonged FDT of 15,36,or 50 minutes were higher than those simulated using conventional EBRT.The late-responding normal tissue cell line HEI-OC1 with a ?/? ratio of 1.096 was more sensitive to the prolonged FDTs than acute-responding normal tissue cell line HaCaT with a ?/? ratio of 9.83.To determine whether prolonged fraction delivery times affected the production and repair of DNA double-strand breaks(DSBs)induced by irradiation,cells irradiated with different FDTs were examined for radiation-induced foci(RIF)formation using y-H2AX immunofluorescence.Compared to HaCaT cells,HEI-OC1 cells showed a significant decrease in y-H2AX foci formation with prolonged FDT.To further study the effect of prolonged fraction delivery time on apoptosis of HEI-OC1 and HaCaT cells,apoptosis of both cells was measured using flow cytometry with the Annexin V/PI apoptosis detection kit.Groups exposed to a prolonged FDT of 16,37,and 51 minutes showed a decrease in the percentage of apoptotic cells compared to the group with a FDT of 2 minutes for both cells.However,this reduction in apoptosis was more obvious in HEI-OC1 cells compared to HaCaT cells.Taken together,these results suggested that the late-responding normal tissue cell line HEI-OC1 was more sensitive to radiation with prolonged FDTs than early-responding normal tissue cell line HaCaT.2.Prolonged fraction delivery time engages the ATM/AMPK signaling pathway to induce autophagy in HEI-OC1To explore the mechanism underlying the different responses to prolonged FDTs between HEI-OC1 and HaCaT cells,we examined the autophagic activities in these two cell lines.LC3,p62 and Beclinl proteins were analyzed to assess the effect of different irradiation protocols on the autophagic pathway.In HEI-OC1 cells,prolonged FDT increased the levels of LC3-II and Beclinl and decreased the level of p62 compared to a FDT of 2 minutes,while prolonged FDTs did not induce different autophagic activity in HaCaT cells.In addition,a GFP-LC3 assay showd that prolonged FDTs resulted in an enhanced accumulation of GFP-LC3 puncta in HEI-OC1 cells but not in HaCaT cells.Ataxia-telangiectasia mutated(ATM)and AMP-activated protein kinase(AMPK)have been reported to activate and regulate autophagy in cells exposed to stress.Thus,we investigated ATM and AMPK signaling in HEI-OC1 cells after irradiation.Exposure of HEI-OC1 cells to IR with prolonged FDT activated ATM and AMPK signaling,and treatment with the ATM inhibitor KU55933 at a concentration of 100 nM decreased levels of P-ATM and P-AMPK.Interestingly,the enhanced autophagic activity induced by prolonged FDTs was attenuated after inhibition of the ATM signal.Taken together,these results suggested that prolonged FDTs engages the ATM/AMPK signaling pathway to induce enhanced autophagic activity in HEI-OC1 cells,but not in HaCaT cells.3.Autophagy contributes to the protective effect induced by prolonged FDT on HEI-OC1 cells via ROS deletionTo investigate the relationship of increased autophagy caused by prolonged FDTs in HEI-OC1 and the enhanced sensitivity of late-responding normal tissue cell to prolonged FDTs,we examined whether modulation of the autophagic pathway affected cell survival in cells treated with different irradiation protocols.We analyzed the effects of the autophagy inhibitor 3-MA and ATM inhibitor KU55933 on clonogenic survival in different groups.After treatment with 3-MA or KU55933,protective effect induced by prolonged FDT was attenuated in HEI-OC1 cells.We also detected the intracellular ROS level using fluorescent probe dichlorofluorescin diacetate(DCFH-DA).Irradiation increased ROS levels in HEI-OC1 cells,and when exposed to radiation with prolonged FDT,the intracellular ROS level decreased.After treatment with 3-MA or KU55933,the intracellular ROS level increased and the reduction in the ROS level induced by prolonged FDTs disappeared.These results indicated that autophagy induced by prolonged FDTs protects HEI-OC1 cells from irradiation injury via a decrease in ROS.Conclusion:Prolonged FDTs protect normal tissue cells from radiation damage,and these effects may be strengthened with more prolonged FDTs.Late-responding normal tissue demonstrated a greater benefit from changes in fraction delivery time compared to acute-responding tissue because prolonged FDTs induced a stronger cytoprotective autophagy by engaging the ATM/AMPK signaling pathway.