EFFECT OF RADIATION ON THE SENSORI-NEURAL AUDITORY SYSTEM AND THE CLINICAL IMPLICATIONS LOW WONG KEIN CHRISTOPHER (MBBS, FRCSEd, FRCSGlas, FAMS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF OTOLARYNGOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2006 i Dedicated to: My wife Stephanie Lim Yin Chan, whose sacrifice, patience, encouragement and love, had been major contributions to the success of this thesis. My colleague Goh Lip Kai, whose valuable contributions are greatly missed as a result of his untimely demise. ii ACKNOWLEDGMENTS I am very grateful to the following individuals and appreciate their contributions to the success of this thesis: 1. Supervisor A/Prof Wang De-Yun for his sound advice, kind guidance, constant encouragement and facilitation of funding. 2. Prof Peter Alberti, Dr Ruan Runsheng, A/Prof Mary Veronique Clement and A/Prof Bay Boon Huat for their constructive advice 3. Prof Yeoh Kian Hian and Prof Tay Boon Keng for their support in my application for admission to this course 4. Dr Michelle GK Tan, Dr Sun Li, Mr Alvin Chua, Mr Goh Lip-Kai, Dr Julian Wee, Dr Toh Song Tar, Dr Fong Kam Weng, Mr Ronald Burgess, Ms Stephanie Fook Cheong and Mr JS Tay for having assisted in the laboratories, clinics or publications. 5. Dr F. Kalinec from the House Ear Institute, USA who had very generously donated the OC-k3 cell line 6. Prof S. Nagata, University of Osaka, Japan, for his consent to the use his illustration 7. Staff from the Dept of Otolaryngology, Singapore General Hospital and Department of Therapeutic Radiology, National Cancer Centre who had facilitated the studies on patients. The studies had been partially supported by grants from National Medical Research Council of Singapore, from the Department of Clinical Research, Singapore General Hospital and from the National University of Singapore. iii PUBLICATIONS Low WK, Burgess R, Fong KW, Wang DY. Effect of radiotherapy on retrocochlear auditory pathways. Laryngoscope. 2005 Oct; 115(10) :1823-6 Low WK, Toh ST, Wee J, Fook-Chong SM, Wang DY.Sensorineural hearing loss after radiotherapy and chemoradiotherapy: a single, blinded, randomized study. J Clin Oncol. 2006 Apr 20;24(12):1904-9 Low WK, Tan MG, Sun L, Chua AW, Goh LK, Wang DY Dose-dependant radiation-induced apoptosis in a cochlear cell-line. Apoptosis 2006; 11:2127-36 iv TABLE OF CONTENTS Acknowledgments Summary List of Tables List of Figures List of abbreviations 1. Introduction ………………………………………………………………………….1 2. Literature Review …………………………………………………………………….4 2.1 Characteristics of radiation-induced sensori-neural hearing loss 2.1.1 Animal studies 2.1.2 Human studies 2.1.3 Prevalence and epidemiology 2.1.4 Effect of radiation dose 2.1.5 High frequency hearing loss 2.1.6 Early vs late onset 2.2 Effect of radiation on retro-cochlear auditory pathways 2.2.1 The sensori-neural auditory pathways 2.2.2 Mechanisms of damage to the nervous system 2.2.3 Cochlear vs retro-cochlear damage 2.2.4 Cerebral and brainstem damage v 2.2.5 Spiral ganglia and cochlear nerve damage 2.2.6 Necropsy studies 2.2.7 Clinical studies 2.2.8 Gamma radiosurgery for acoustic neuroma 2.3 Combined effects of radiation and cisplatin on sensori-neural hearing 2.3.1 Combined chemo-radiotherapy for head and neck tumours 2.3.2 Properties of cisplatin 2.3.3 Combined ototoxicity of radiation and cisplatin 2.4 Cellular and molecular basis of ototoxicity 2.4.1 Apoptotic cell death 2.4.2 Necrotic cell death 2.4.3 Apoptosis in the cochlea 2.4.3.1 Ototoxicity 2.4.3.2 Caspases 2.4.3.3 Bcl-2 family 2.4.3.4 p53 protein 2.4.3.5 c-jun NH2-terminal pathway 2.4.3.6 Reactive oxygen species 2.5 Radiation-induced apoptosis 2.5.1 Radiobiology vi 2.5.2 Radiation-induced cell death 2.5.3 Cellular targets of radiation 2.6 Intervention strategies based on cellular and molecular mechanisms 2.6.1 Protection against apoptosis 2.6.2 L-Acetylcysteine 2.6.3 Hair cell regeneration 2.6.3.1 Strategies 2.6.3.2 Genetic manipulation in the mammalian inner ear 2.6.3.3 Gene therapy in the mammalian cochlea 2.6.3.4 Stem cells in the inner ear 2.7 Using cochlear cell lines to study ototoxicity 2.7.1 Difficulty in studying the basic mechanisms of ototoxicity 2.7.2 Use of cell lines 2.7.3 Cell lines based on the transgenic mice 2.7.4 Cochlear cell lines from the transgenic mice 2.8 Summary of literature review 3. Objectives …………………………………………………………………………55 4. To study the effect of radiation on retro-cochlear pathways ………………………57 4.1 Abstract 4.2 Background 4.3 Material & Methods 4.3.1 Patients vii 4.3.2 Audiological measurements 4.3.3 Radiotherapy technique 4.3.4 Radiation dose measurements 4.4 Results 4.4.1 Audiological effects of radiation 4.4.2 Radiation doses to the cochlea and the cochlear nerve 4.5 Comments 5. To study the synergistic ototoxic effects of radiation and cisplatin …………………76 5.1 Abstract 5.2 Background 5.3 Material & Methods 5.3.1 Patients 5.3.2 Treatment 5.3.3 Randomisation 5.3.4 Inclusion criteria 5.3.5 Audiological assessment 5.3.6 Statistical methods 5.4 Results 5.4.1 Analysis across groups 5.4.2 Analysis within each group 5.5 Comments viii 6. To study radiation-induced apoptosis in a cochlear cell line………………………96 6.1 Abstract 6.2 Background 6.3 Materials & Methods 6.3.1 Cell culture 6.3.2 Cell viability assay 6.3.3 Apoptotic analysis 6.3.3.1 Flow cytometry 6.3.3.2 TUNEL assay 6.3.4 Microarray analysis 6.3.5 Western blot analysis 6.3.6 Measurement of intra-cellular reactive oxygen species generation 6.3.7 Statistical analysis 6.4 Results 6.4.1 Post-irradiation viability of cells is dose related 6.4.2 Dose-related apoptosis occurs predominantly at 72 hrs 6.4.3 Generation of reactive oxygen species is dose-related 6.4.4 Gene changes involved in apoptosis are dose related 6.4.5 Effect of radiation on p53 and c-jun 6.5 Comments 7. To study the protective effects of anti-oxidant L-NAC ……………………………122 7.1 Abstract ix 7.2 Background 7.3 Material & Methods 7.3.1 Cell culture 7.3.2 Cell viability assay 7.3.3 Flow cytometry 7.3.4 TUNEL assay 7.3.5 Confocal microscopic and fluorescence intensity analysis 7.3.6 Data analysis 7.4 Results 7.4.1 L-NAC cell viability 7.4.2 L-NAC protects against radiation-induced apoptosis 7.4.3 L-NAC inhibits ROS generation 7.5 Comments 8. Discussion .……………………………………………………………………….138 8.1 Radiation-induced SNHL in doses used clinically is an intra-cochlear event 8.2 Damage preferentially occurs in the basal coil of the cochlea 8.3 Radiation-induced SNHL based on an apoptotic model 8.3.1 A p53-dependent apoptotic model 8.3.2 A ROS-related apoptotic model 8.4 Radiation-induced SNHL based on multiple cell death mechanisms 9. Conclusion…………………………………………………………………………153 x 10. Bibliography ……………………………………………………………………. 155 xi SUMMARY Irradiation-induced sensori-neural hearing loss (SNHL) is common after radiotherapy (RT) to head and neck tumours, and yet the cellular and molecular mechanisms leading to SNHL remain unclear. This problem is especially relevant in Singapore because nasopharyngeal carcinoma (NPC) is common and RT is the main modality of treatment for this condition. In this thesis, a prospective study on NPC patients treated by RT was carried out. It confirmed that the auditory pathways received relatively high radiation doses but the retro-cochlear pathways measured by brainstem evoked response audiometry (BERA), remained functionally intact even up to years post-radiation. This suggests that the site of lesion in radiation-induced SNHL is at the cochlear and not the retro-cochlear level. Combined chemo-radiation therapy using cisplatin (CDDP) is increasingly used in head and neck cancers and CDDP is well known to be also ototoxic. A single-blinded randomized study on NPC patients treated with RT alone and with chemo-radiation therapy was carried out over a 2-year period. Combined therapy resulted in a synergistic ototoxic effect, particularly for high frequency sounds in the speech range. Based on a tonotopic arrangement of hair cells in the cochlea, the observed patterns of hearing loss suggested preferential basal-to-apical hair cell destruction in the cochlea. This could be related to mechanisms involving reactive oxygen species (ROS), as the concentration of ROS in cochlear hair cells is also known to follow a basal-toapical pattern. Although it is known that ROS-induced apoptosis of cochlear cells occurs in aminoglycoside and CDDP-induced ototoxicity, apoptotic cochlear cell death has not been studied in radiation-induced ototoxicity. In this thesis, radiation-induced apoptosis xii was confirmed by flow cytometry and TUNEL assay in a cochlear cell line (OC-k3). The post-irradiation gene expression profiles were analysed by microarray studies and proteins resulting from selected up-regulated genes were further investigated by Western blotting. Intracellular generation of ROS was detected by 2’, 7’-dichlorofluorescein diacetate (DCFDA), with comparisons made using confocal microscopy and fluorescence intensity. It was found that radiation-induced apoptosis occurred in a dose-dependant and ROS-related fashion with p53 possibly playing a major role. The anti-oxidant L-NAcetylcysteine (L-NAC) protected the cell line against radiation-induced apoptosis. This thesis supports the feasibility of cochlear implantation, if one is clinically indicated, for patients whose auditory pathways had previously been irradiated. For a successful outcome after cochlear implantation, it is a prerequisite for the retro-cochlear pathways to be intact. As the thesis confirmed that patients who had received RT and concurrent/adjuvant chemotherapy experienced greater sensori-neural hearing loss compared to those treated by RT alone, normal inner ear tissue tolerance once defined only for radiotherapy alone should be redefined in chemo-radiotherapy. Based on a ROSrelated apoptotic model, there is the possibility of preventive strategies targeted at different stages of the apoptotic process. Although multiple cell death mechanisms may be involved in radiation-induced SNHL, they are likely to share common upstream signaling process such as ROS generation. Therefore, the use of L-NAC in protecting against radiation-induced SNHL clinically, looks promising. Not only has it already been safely used in humans for other clinical indications, large doses can potentially be administered topically through the middle ear with minimal systemic side effects. xiii LIST OF TABLES Table Table Title Page Evoked response audiometric inter-wave latencies before, during and after radiotherapy. 67 Pre- and post-radiotherapy pure-tone audiogram bone-conduction hearing levels in ears which recorded significant sensori-neural hearing deterioration after treatment. 68 Descriptive statistics of the minimum, maximum and mean point doses delivered to each side of the cochlea and the internal auditory meatus. 69 Dose schedules for concurrent chemo-radiotherapy and adjuvant chemotherapy 82 Table Characteristics of patients in each treatment group 84 Table Bone-conduction thresholds at the high and lower speech frequencies for patients in the radiotherapy and chemo-radiotherapy groups at different post-radiotherapy time-points 87 Table Table Table Table An overview of the biological features of genes regulated by gamma-irradiation in OC-k3 cells 115 xiv LIST OF FIGURES Figure Title Page Figure Signal transduction for apoptosis 26 Figure Beam's Eye View of lateral the post-nasal space field 63 Figure Axial CT scan of the nasopharynx showing a computer generated isodose plan 64 Box-plots to compare pre-treatment median sensori-neural hearing thresholds in the lower speech frequencies with the corresponding values at different post-radiotherapy time-points for patients in the radiotherapy and chemo-radiotherapy groups 88 Box-plots to compare pre-treatment median sensori-neural hearing thresholds at 4kHz with the corresponding values at different post-radiotherapy time-points for patients in the radiotherapy and chemo-radiotherapy groups 89 Post-irradiation viability of cells is dose-related and MTT absorbance values for different radiation doses at various time-points are shown 109 Flow cytometry to show the effect of radiation dose on cell cycle phase distribution 110 TUNEL assay confirms irradiation-induced apoptosis in OC-k3 cells at 72 hrs and is dose related 111 Figure Figure Figure Figure Figure xv Figure Radiation-induced generation of ROS is dose related 112 Figure 10 Overview of gene changes at 24 and 72 hrs after and 20 Gy of radiation 113 Western blot showing p53 and c-jun protein expression and phosphorylation at various time-points after 0, and 20Gy of radiation 114 Figure 11 Figure 12 MTT assay shows post-irradiated OC-k3 cell viability is increased by L-NAC 130 Figure 13 Flow cytometry showing the effect of L-NAC on the cell cycle phase distribution in γ-irradiated OC-k3 cells 131 TUNEL assay confirms L-NAC protects against radiation-induced apoptosis 132 L-NAC reduces intracellular ROS generated by γ-irradiation 133 Figure 14 Figure 15 xvi LIST OF ABBREVIATIONS BERA Brainstem evoked response audiometry CDDP Cisplatin DCFDA 2’, 7’-dichlorofluorescein diacetate GSH Reduced Gluthathione Gy Gray IAM Internal Auditory Meatus L-NAC L-N-Acetylcysteine MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide NPC Nasopharyngeal carcinoma OHC Outer hair cells ROS Reactive oxygen species RT Radiotherapy SNHL Sensori-neural hearing loss TUNEL Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling xvii [...]... after 5 and 20 Gy of radiation 11 3 Western blot showing p53 and c-jun protein expression and phosphorylation at various time-points after 0, 5 and 20Gy of radiation 11 4 Figure 11 Figure 12 MTT assay shows post-irradiated OC-k3 cell viability is increased by L-NAC 13 0 Figure 13 Flow cytometry showing the effect of L-NAC on the cell cycle phase distribution in γ-irradiated OC-k3 cells 13 1 TUNEL assay confirms... different radiation doses at various time-points are shown 10 9 Flow cytometry to show the effect of radiation dose on cell cycle phase distribution 11 0 TUNEL assay confirms irradiation-induced apoptosis in OC-k3 cells at 72 hrs and is dose related 11 1 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 xv Figure 9 Radiation- induced generation of ROS is dose related 11 2 Figure 10 Overview of gene changes at 24 and. .. Table 1 Title Page Evoked response audiometric inter-wave latencies before, during and after radiotherapy 67 Pre- and post-radiotherapy pure-tone audiogram bone-conduction hearing levels in ears which recorded significant sensori- neural hearing deterioration after treatment 68 Descriptive statistics of the minimum, maximum and mean point doses delivered to each side of the cochlea and the internal auditory. .. condition In this thesis, a prospective study on NPC patients treated by RT was carried out It confirmed that the auditory pathways received relatively high radiation doses but the retro-cochlear pathways measured by brainstem evoked response audiometry (BERA), remained functionally intact even up to 4 years post -radiation This suggests that the site of lesion in radiation- induced SNHL is at the cochlear... schedules for concurrent chemo-radiotherapy and adjuvant chemotherapy 82 Table 5 Characteristics of patients in each treatment group 84 Table 6 Bone-conduction thresholds at the high and lower speech frequencies for patients in the radiotherapy and chemo-radiotherapy groups at different post-radiotherapy time-points 87 Table 2 Table 3 Table 4 Table 7 An overview of the biological features of genes regulated... different post-radiotherapy time-points for patients in the radiotherapy and chemo-radiotherapy groups 88 Box-plots to compare pre-treatment median sensori- neural hearing thresholds at 4kHz with the corresponding values at different post-radiotherapy time-points for patients in the radiotherapy and chemo-radiotherapy groups 89 Post-irradiation viability of cells is dose-related and MTT absorbance values... SNHL, they are likely to share common upstream signaling process such as ROS generation Therefore, the use of L-NAC in protecting against radiation- induced SNHL clinically, looks promising Not only has it already been safely used in humans for other clinical indications, large doses can potentially be administered topically through the middle ear with minimal systemic side effects xiii LIST OF TABLES.. .10 Bibliography …………………………………………………………………… 15 5 xi SUMMARY Irradiation-induced sensori- neural hearing loss (SNHL) is common after radiotherapy (RT) to head and neck tumours, and yet the cellular and molecular mechanisms leading to SNHL remain unclear This problem is especially relevant in Singapore because nasopharyngeal carcinoma (NPC) is common and RT is the main modality of treatment for this condition... against radiation- induced apoptosis This thesis supports the feasibility of cochlear implantation, if one is clinically indicated, for patients whose auditory pathways had previously been irradiated For a successful outcome after cochlear implantation, it is a prerequisite for the retro-cochlear pathways to be intact As the thesis confirmed that patients who had received RT and concurrent/adjuvant chemotherapy... greater sensori- neural hearing loss compared to those treated by RT alone, normal inner ear tissue tolerance once defined only for radiotherapy alone should be redefined in chemo-radiotherapy Based on a ROSrelated apoptotic model, there is the possibility of preventive strategies targeted at different stages of the apoptotic process Although multiple cell death mechanisms may be involved in radiation- induced . and is dose related 11 1 xvi Figure 9 Radiation- induced generation of ROS is dose related 11 2 Figure 10 Overview of gene changes at 24 and 72 hrs after 5 and 20 Gy of radiation 11 3. EFFECT OF RADIATION ON THE SENSORI-NEURAL AUDITORY SYSTEM AND THE CLINICAL IMPLICATIONS LOW WONG KEIN CHRISTOPHER (MBBS, FRCSEd, FRCSGlas, FAMS) A THESIS SUBMITTED FOR THE. …………………………………………………………………….4 2 .1 Characteristics of radiation- induced sensori-neural hearing loss 2 .1. 1 Animal studies 2 .1. 2 Human studies 2 .1. 3 Prevalence and epidemiology 2 .1. 4 Effect of radiation dose 2 .1. 5 High