Establishment and characterization of radiation resistant strains from squamous cell carcinoma cell lines in serum free defined culture

Ph.D THESIS Establishment and characterization of radiation resistant strains from squamous cell carcinoma cell lines in serum-free defined culture by NGUYEN QUANG TAM Department of Molecular Oral Medicine and Maxillofacial Surgery Graduate School of Biomedical & Health Sciences Hiroshima University 2018 ACKNOWLEDGEMENT Foremost, I would like to express my deep gratitude to my Academic Supervisor Professor Tetsuji Okamoto, who has accepted me to study in the department of Molecular Oral Medicine and Maxillofacial Surgery and also in Phoenix Education Leading Program of Hiroshima University He always finds out my mistakes and teach me how to fix in a better way I have learned a lot from him that I must try my best in doing scientific research and never give up, also be creative in thinking I would also like to thank Professor Chisa Shukunami, Professor Shinya Matsuura, Professor Satoru Endo and Professor Nobuyuki Chikudate for being my great co-advisers during my study My deep thank is also extended to Professor Naoya Kakimoto, Professor Masaru Sugiyama and Assoc Professor Shigeaki Toratani for reviewing my thesis and their great comments I would like to thank my instructor Dr Hamada Atsuko for teaching me all molecular techniques in my research and help me to analyze the data I would like to thank all Associate and Assistant Professors in my lab, all my seniors, my lab-mates for their supports in my research and other activities My grateful thanks are also extended to Assoc Prof Tran Diep Tuan, Assoc Prof Ngo Thi Quynh Lan, and Dr Luong Van To My for introducing me to study in Hiroshima University and my colleagues, who are covering my works in Vietnam Finally, I would like to extend my utmost gratitude to my family for their great encouragement when I study in Japan TABLE OF CONTENTS Summary Chapter 1: Introduction …………………………………………………………………….1 Chapter 2: Establishment and characterization of radiation resistant strains …………3 I Materials and methods .3 Cell culture & culture medium Irradiation procedure for generation of radiation resistant (RR) strains Colony survival assay for wild type (WT) and radiation resistant strains Growth rate of WT and RR- strains Sphere formation assay of WT and RR- strains Expression of cancer stem cell marker CD133 in WT and RR- strains Expression of pluripotent stem cell markers Nanog, Oct4, and Sox2 in WT and RR- strains by Real time-quantitative polymerase chain reaction (RTqPCR) Migration assay of WT and RR- strains Tumor formation ability of WT and RR- strains in nude mice 10 Immunohistochemical examination of ki67 in nude mouse tumors derived from WT and RR- strains 11 Statistical analysis II Results Establishment of radiation resistant cancer cell strains and colony survival formation Proliferation of the cells in serum-free monolayer culture Sphere forming ability of the cells in serum-free suspension culture Expression of cancer and pluripotent stem cell markers in WT and RRstrains Migration ability of WT and RR- strains Tumor formation ability of WT and RR-strains in nude mice Immunohistochemical expression of ki67 in nude mice tumors derived from WT and RR-strains Chapter 3: Identification and characterization of novel genes involved in radiation resistance … 12 I Materials and methods 12 DNA microarray analysis Expression of IGF2 and krt13 in wild type and radiation resistant strains by RT-qPCR Western-blotting Immunohistochemical expression of IGF2 and KRT13 proteins in nude mice tumors derived from WT and RR-strains II Results 13 Results of DNA microarray analysis Expression of IGF2 gene and protein in WT and RR-strains by RT-qPCR, western-blotting and immunohistochemical staining Expression of krt13 gene and protein in WT and RR-strains by RT-qPCR, western-blotting and immunohistochemical staining Chapter 4: Functional analysis of IGF2 and krt13 in radiation resistance 16 I Materials and methods 16 Effect of krt13 siRNA and IGF2 siRNA transduction on colony formation ability of RR-strains Effect of radiation on IGF2-siRNA transduced-A431-LDR, -HDR, NALDR and -HDR cells Effect of radiation of krt13-siRNA transduced-A431-LDR and -HDR cells Expression of pluripotent stem cell markers, krt13 and IGF2 in IGF2- and krt13-siRNA transduced cells Generation of stable transfectant of krt13 in A431 cells Characterization of krt13-transfected A431 cell and its radiation resistant ability II Results 19 Effect of various IGF2- and krt13-siRNA on silencing ability in the cells Effect of radiation on colony survival formation of IGF2-siRNA transduced A431-LDR, -HDR, NA-LDR and -HDR cells Effect of radiation on colony survival formation of krt13-siRNA transduced A431-LDR, -HDR cells 4 Expression of krt13, IGF2 and pluripotent stem cell marker genes in IGF2siRNA transduced A431-LDR Expression of IGF2, krt13 and pluripotent stem cell marker genes in krt13siRNA transduced A431-LDR Characterization of A431 cells overexpressing krt13 in serum-free culture Chapter 5: Discussion 21 Chapter 6: Conclusion 27 References 29 Figure legends 36 ABBREVIATIONS A431-HDR A431-LDR A431-WT CSCs DMEM EDTA GAPDH HDR IGF2 Jak/STAT kDa krt13 LDR MAPK mRNA NA-HDR NA-LDR NA-WT OSCC PBS PVDF RR RR-SCC RT RT-qPCR SCC siNC siRNA WT A431 radiation resistant cells established in high dose rate irradiation A431 radiation resistant cells established in low dose rate irradiation A431 wild type cells cancer stem cells Dulbecco’s modified Eagle’s medium Ethylene Diamine Tetra Acetic acid Glyceraldehyde-3-phosphate dehydrogenase high dose rate insulin-like growth factor Janus kinase/signal transducer of activation kilo Dalton keratin 13 low dose rate mitogen-activated protein kinase messenger RNA NA radiation resistant cell established in high dose rate irradiation NA radiation resistant cell established in low dose rate irradiation NA wild type cell oral squamous cell carcinoma phosphate-buffered saline polyvinylidene difluoride radiation resistant radiation resistant squamous cell carcinoma radiation therapy Reverse Transcription-quantitative Polymerase Chain Reaction squamous cell carcinoma cells transfected with negative control siRNA small interfering RNA wild type Summary Introduction Squamous cell carcinoma (SCC), including oral squamous cell carcinoma (OSCC), has been increasing in the world and being the most common cancer in South-East Asian countries where have a betel-quid and areca-nut chewing habit It has been considered that cancer cells are functionally heterogeneous that undergo not only proliferation but also differentiation and maturation to a certain degree and contain a small population of cancer stem cells (CSCs) It seems logical that cures of cancer can be achieved only if the CSCs population is eliminated Several treatment modalities such as operation, radiation, and chemo therapy have been reported to be effective in treating many kinds of cancer including OSCC Among them, radiation therapy (RT) plays a major role in the management of OSCC Despite therapeutic and technological advances such as ionizing radiation, gamma rays and charged particles to kill cancer cells through DNA damage directly or indirectly caused by free ion radicals, some patients will have persistence of irradiated tumor or develop locoregional failure, resulting in significant morbidity and mortality RT using high dose rate (HDR) radiation has been widely used as an effective modality to treat human cancer by various types of modern delivering techniques On the other hand, RT using low dose rate (LDR) radiation has been introduced in the treatment of prostate and oral cancers Therefore, to elucidate the cellular and molecular mechanisms involved in radiation resistance of cancer cells upon radiation therapy using HDR or LDR might be worthwhile to develop effective therapies to circumvent radiation resistance In this work, mechanisms of radio-resistance to RT in squamous cell carcinomas including oral SCC and strategies used to overcome this resistance were studied To study that, it might be useful to establish radiation resistant-cancer cells in in vitro Although there have been several reports of isolating radiation resistant cancer cells, and their use to elucidate cellular and molecular mechanisms in radiation resistance, these radiation resistant (RR) SCC cells were isolated under serum-supplemented culture condition Serum-supplemented medium contains a lot of undefined or unknown proteins, factors, and lipids which may exhibit unknown biological effects on cancer cells in vitro Thus, serum-free defined medium can show exact biological characteristic of the cells In this study, first I have tried to isolate and establish RR-SCC strains from SCC and OSCC cell lines in serum-free defined culture, and then characterized their cellular and molecular properties, and defined functional genes involved in radiation resistance Materials and methods Two SCC cell lines, A431 derived from vulvar SCC, and NA/HO-1-N-1 from OSCC were used in this study The cells were cultured in serum-free DF6F medium (1:1 mixture of Dulbecco’s Modified Eagle Media and Ham-F12 medium supplemented with insulin (10 g/ml), transferrin (5 g/ml), 2-aminoethanol (10 M), sodium selenite (10 nM), 2mercaptoethanol (10 M), and oleic acid conjugated with fatty acid-free bovine serum albumin (9.4 g/ml)) All cell lines were irradiated weekly at a dose of 2.2Gy/day, days/week with a low dose rate (LDR) irradiation system (RM1000, Chugai Technos, Japan), or at 5Gy/5.75 mins, twice a week with a high dose rate (HDR) system (Gamma cell 40 Exactor, Best Theratronics, Canada) in serum-free defined culture After irradiating under 60Gy as a whole dose by LDR and HDR irradiation system, we have isolated RR sub-strains from SCC cell lines To confirm the radiation resistance of these RR-strains, colony survival assay was performed as follow The wild type (WT) and RR strains were irradiated at a dose of 0Gy, 2Gy, 4Gy, 6Gy, and 8Gy, respectively, and examined radiation resistance by colony survival assay After 14 days of culture, the colonies were stained with Giemsa, and counted To clarify the biological properties of these cells, several cellular abilities such as growth in monolayer culture, sphere formation in suspension culture, and migration in Boydenchamber method were examined in serum-free culture For the growth assay, the cells were seeded at 104cells/well in 24-well plate and counted cell numbers every day by the Coulter Counter For sphere formation assay, the cells were seeded in 35mm prime surface (lowattachment) dish at 103cells/dish, and then sphere numbers were counted on day For migration assay, the cells were seeded at 5x104cells/well in 24-well collagen coated chemotaxicell well in DF medium supplemented with 0.1% BSA and stained with Giemsa for counting number of migrated cells/mm2 The ratio of CD133 positive cell in each cell line was examined by flowcytometry as cancer stem cell marker Total RNAs extracted and purified from all cell lines, were used for Real Timequantitative PCR (RT-qPCR), and DNA microarray analysis Expression of pluripotent stem cell markers Nanog, Oct4 and Sox2 in WT and RR-strains in WT and RR-strains was examined with RT-qPCR To study the tumor forming ability in nude mouse, WT and RR-strains (0.25 x106 -1x106 cells/100l DF) were injected subcutaneously to the dorsal back skin of nude mice (BALB/cnu/nu), and tumor size was measured every week Then the tumors were excised, weighted, fixed in 4% paraformaldehyde for 24hr, and embedded in paraffin for H&E and immunohistochemical staining DNA microarray analysis of all cells was conducted for further investigation In DNA microarray data, IGF2 and krt13 are highly expressed in RR-strains compare to WT among and were chosen for further investigation Theirs high expression in RR-strains in RNA level and protein level were confirmed by RT-qPCR analysis, western blot and immunohistochemical staining of nude mice tumors Silencing of those genes in RR-strains by siRNA were also conducted to confirm their relations with radiation resistance The function of the IGF2 and krt13 in RR-strains were studied by silencing with siRNA and overexpression by generation of stable transfectant krt13-A431 cell for checking their radiation sensitivity, pluripotent stem cell marker expression, growth in monolayer and sphere forming ability Results By using LDR and HDR system, RR-strains from A431-WT and NA-WT cells designated A431-LDR, NA-LDR, A431-HDR, and NA-HDR, were successfully isolated in serum-free defined culture The D37 value of A431-LDR, A431-HDR, A431-WT, NA-LDR, NA-HDR, and NA-WT was 5Gy, 3.7Gy, 2.3Gy, 7.5Gy, 5.5Gy and 4.6Gy, respectively These cells exhibited higher expression of cancer stem cell marker such as CD133, higher sphere formation and higher migration abilities compare to those of WT cell lines LDR-RR cells showed significant higher expression of pluripotent stem cell marker Nanog than in WT and HDR-RR cells In addition, the RR cells exhibited higher tumor forming ability compared to WT cells in nude mice xenograft LDR radiation can generate higher radiation resistance of cancer cells, higher expression of Nanog, higher migration ability and tumor forming ability than in HDR system DNA microarray analysis revealed over 500 genes were overexpressed in RR-strains compared to WT cells Among them, IGF2 and krt13 genes were high expression in RR-strains compare to WT cells RT-qPCR analysis further confirmed that both RR-strains overexpressed IGF2 and krt13 In addition, pathway analysis of the DNA microarray analysis revealed that various pathways, such as MAPK, JAK/STAT signaling pathway, apoptosis pathway, TGF- signaling pathway and cytokine-cytokine receptor interaction were activated in RR-strains Gene Ontology analysis of microarray analysis also showed that the genes involved in keratinization, inflammatory response, wound healing and response to cytokine stimulus were enriched 10 (A) (B) Figure 5: Expression of cancer stem cell marker CD133 in WT and RR-strains 47 (A) (B) Figure 6: Expression of pluripotent stem cell markers Nanog, Oct4, Sox2 in WT and RR-strains (*p