학술논문
Omics-based identification of novel molecular signaling networks associated with low and high-LET radiation in tumor mimic spheroid and rodent model
Document Type
Dissertation/ Thesis
Author
Source
Subject
Language
English
Abstract
Although conventional clinical treatment with low-LET (linear energy transfer), such as gamma-ray and X-ray, has been widely used for radiotherapy in various cancers, ineffective outcomes occur due to radioresistance. High-LET has become an alternative treatment option since it is able to induce apoptosis. Indeed, a critical study has shown that high-LET triggered high effectiveness of apoptosis in human cancer. However, the molecular mechanisms toward high-LET have not been clarified yet. Furthermore, most studies associated with high-LET have been carried out in monolayer culture systems, which less-accurately represent solid tumor microenvironment than multicultural spheroid systems. Here we applied in vivo mimicing 3D spheroid system to investigate molecular signaling networks triggered by high-LET neutron irradiation. Also, we applied high-throughput techniques, such as array comparative genomic hybridization, gene expression microarray, and 2-dimensional gel electrophoresis to investigate molecular events triggered by neutron. As a result, a 3D spheroid system was achieved using a thermoreversible gel system. An effective apoptosis-inducible dose of neutron was determined by Acridine Orange (AO) staining in 3D spheroid. Our data showed a radioresistant phenotype in p53-deficient MCS relative to p53-proficient MCS. However, we also observed a certain level of apoptotic index in p53-deficient MCS under neutron exposure. We found a few copy number gains and losses in p53-profieicnt and -deficient MCS following neutron exposure. Based on gene expression profiles, we identified novel targets and signaling networks associated with neutron-induced apoptosis in MCS. In addition, several protein target genes were also discovered via 2-dimensional gel electrophoresis. In conclusion, we demonstrated novel molecular signaling and corresponding targets of in vitro solid tumors following high-LET exposure at DNA, mRNA, and protein levels. This result provides critical progression towards a clarification of neutron-induced apoptosis mechanisms.
Ionizing radiation (IR) therapy using gamma-ray is a potential treatment option of several solid tumor forms; however, the molecular responses of carcinoma cells to IR are not fully understood. A multicellular spheroid (MCS)-in vivo mimic model was established allow us to study cellular responses to gamma-rays in human carcinoma cells in a closer representation of the in situ environment. Here, relative cell survival rate was determined in p53-wild and-null type cells of MCS in comparison to that of monolayer culture in response to 10 Gy gamma-ray. Trypan blue exclusion assay showed that under MCS culture systems a radioresistance phenotype was dependent on p53 status since p53-null type MCS exhibited a significantly higher cell survival rate, than p53-wild type MCS. In order to analyze the molecular signaling pathway regarding gamma-ray-induced apoptosis, we conducted microarray under 10 Gy of gamma-ray to observe the gene expression pattern change in MCS compared to monolayer cells, with and without p53. In addition, the potential molecular network was analyzed using Pathway Studio software, to define responsive signaling and interactions. We found novel target genes and signaling networks associated with gamma-ray-triggered apoptosis in p53-proficient and -deficient multicellular spheroid systems. Several genes including IL8, CXCL1, ANXA1, and OSCAR were recognized as key regulators in p53-related apoptosis signaling under MCS. In p53-deficiect MCS culture system, AKT1, MTOR, CDKN1B, and HIF1A were involved in gamma-ray-induced apoptosis signaling. Furthermore, we proposed a mainstream of gamma-ray-triggered apoptosis signaling network for each culture system. A signaling network involving PI3K, AKT, and RAD51C was revealed in monolayer cells, whereas CD38, RAD98, and UBE2V2 participated as main modulators in gamma-ray-triggered apoptosis signaling in the p53-proficient multicellular spheroid system. These genes might be considered as target molecules for evaluation of gamma-ray efficiency in p53-proficient MCS as a typical 3D-in vivo mimic model. Our findings demonstrate the complex and variable responses of p53-carcinoma cells following IR exposure including putative signaling pathways, placing emphasis on the importance of in vivo mimic MCS models rather than conventional monolayer culture systems.
Nanotechnology industries, particularly those using zinc oxide (ZnO), have spurned a rapid growth in the diversity of commercial and widespread products, including cosmetics, food, rubber, paints and plastics, over the last decade. However, in spite of increasing in the population exposure to ZnO, the potential of genotoxicity induced by ZnO has not yet been clarified. To date, there are numerous studies associated with genotoxicity of ZnO, however, the overall verdict is still controversial. The biological effects of nanoparticles can be altered by their physicochemical properties, and within the laboratory, may be altered by the evaluation method used. Thus well-characterized chemicals and appropriate standardized methods are necessarily required for assessing genotoxicity of nanoparticles. In this study, we evaluated the genotoxicity of ZnO using four kinds of well-characterized ZnO nanoparticles: 20 nm negative charge, 20 nm positive charge, 70 nm negative charge, 70 nm positive charge. In vivo alkaline comet assay was conducted following guidelines approved by an international validation study group. Our results showed no statistically significant differences in the toxicity of in vivo comet assay of the four kinds of ZnO nanoparticles, compared to that of the solvent control. The present results suggest that ZnO nanoparticles may not induce genotoxicity, particularly in DNA strand breaks. Furthermore, these results have been generated in accordance with test guidelines, and hence, are considered as reliable information regarding ZnO induced genotoxicity.
Excessive reactive oxygen species (ROS) generation is one of the toxic mechanisms induced by gamma-ray irradiation. Redox factor is a critical player for controlling ROS levels in biosystems, and regulation of this factor by redox stimulators is of critical interest to a great number of areas of medical science. Selenium is well known to have a role in redox regulation and antioxidant function. One of the organic forms of selenium, selenomethionine (SeMet), is relatively nontoxic and can be applied orally to human. In this study, we investigated whether SeMet has a radioprotective effect or not in an animal model. To demonstrate this hypothesis, we conducted in vivo alkaline comet assay and histopathological analysis to determine whether SeMet exerts a radioprotective effect in animal models or not. Furthermore, gene expression profiles and signaling networks were analyzed using gene expression microarray. Also, quantitative real-time PCR were subsequently conducted to validate target genes. We found that gamma-ray-induced DNA damage in mouse spleen with SeMet pretreatment was significantly reduced in comparison to the one treated with gamma-ray alone. Using histopathological analysis, it showed that erythrocyte-containing red pulp was restored in mouse spleen pretreated with SeMet prior to gamma-ray exposure, whereas the red pulp was obviously eliminated in the mouse spleen treated with gamma-ray exposure alone. These results indicate the radioprotective effect of SeMet in mouse tissue, particularly spleen, toward gamma-ray-mediated DNA damage and cell abnormality. We also suggested potential target genes and signaling networks associated with the SeMet-mediated radioprotection mechanism. Our results would provide a critical scientific evidence for the further development of potential radioprotective agents. Furthermore, this information would be advantageous for radiation-related safety management as well as radiotherapeutics in public health fields.
The comet assay can be used to investigate the genotoxicity of various agents, including industrial chemicals, biocides, agrochemicals and pharmaceuticals. It has a number of advantages, such as: sensitivity for detecting low levels of DNA damages, the requirement for small numbers of cells per samples, low costs and ease of application. Nowadays, in vivo comet assay has been suggested as a potential tool for replacement of the in vivo rodent hepatocyte unscheduled DNA synthesis (UDS). International expert groups for comet assay have published recommendations describing standards which are aimed at establishing high quality protocols in order to obtain valid and reliable data. Despite that, international activity for validation of the in vivo comet assay has been carried out, and the distribution of protocol and interlaboratory validation have not yet been conducted in the Republic of Korea. Here, three laboratories participated in an inter-laboratory assessment to test the validity of the in vivo comet assay under the GLP (Good Laboratory Practice) system. Two genotoxic chemicals, N-methyl-N-nitrosourea (MNU) and ethyl methanesulfonate (EMS) were applied, as known and unknown chemicals, respectively, which were selected on the basis of the 3rd international validation study result. Among all GLP laboratories, the results showed a significant increase in DNA damage as % tail DNA as dose-related manner in both liver and stomach tissues treated with MNU relative to the negative control group. Similarly, the EMS-treated group also showed significantly increased % tail DNA damage, in a dose-dependent manner compared with the negative control group in both tissues. The in vivo comet results from all GLP laboratories in Republic of Korea were obviously consistent with the 3rd international validation study. In conclusion, we confirmed the interlaboratory validity of the in vivo comet assay using two genotoxic chemicals, which was assessed by three GLP laboratories in the Republic of Korea. This study can strengthen the in vivo comet assay to be suggested as an international standard protocol or guideline for genotoxicity testing of various genotoxic agents.
Ionizing radiation (IR) therapy using gamma-ray is a potential treatment option of several solid tumor forms; however, the molecular responses of carcinoma cells to IR are not fully understood. A multicellular spheroid (MCS)-in vivo mimic model was established allow us to study cellular responses to gamma-rays in human carcinoma cells in a closer representation of the in situ environment. Here, relative cell survival rate was determined in p53-wild and-null type cells of MCS in comparison to that of monolayer culture in response to 10 Gy gamma-ray. Trypan blue exclusion assay showed that under MCS culture systems a radioresistance phenotype was dependent on p53 status since p53-null type MCS exhibited a significantly higher cell survival rate, than p53-wild type MCS. In order to analyze the molecular signaling pathway regarding gamma-ray-induced apoptosis, we conducted microarray under 10 Gy of gamma-ray to observe the gene expression pattern change in MCS compared to monolayer cells, with and without p53. In addition, the potential molecular network was analyzed using Pathway Studio software, to define responsive signaling and interactions. We found novel target genes and signaling networks associated with gamma-ray-triggered apoptosis in p53-proficient and -deficient multicellular spheroid systems. Several genes including IL8, CXCL1, ANXA1, and OSCAR were recognized as key regulators in p53-related apoptosis signaling under MCS. In p53-deficiect MCS culture system, AKT1, MTOR, CDKN1B, and HIF1A were involved in gamma-ray-induced apoptosis signaling. Furthermore, we proposed a mainstream of gamma-ray-triggered apoptosis signaling network for each culture system. A signaling network involving PI3K, AKT, and RAD51C was revealed in monolayer cells, whereas CD38, RAD98, and UBE2V2 participated as main modulators in gamma-ray-triggered apoptosis signaling in the p53-proficient multicellular spheroid system. These genes might be considered as target molecules for evaluation of gamma-ray efficiency in p53-proficient MCS as a typical 3D-in vivo mimic model. Our findings demonstrate the complex and variable responses of p53-carcinoma cells following IR exposure including putative signaling pathways, placing emphasis on the importance of in vivo mimic MCS models rather than conventional monolayer culture systems.
Nanotechnology industries, particularly those using zinc oxide (ZnO), have spurned a rapid growth in the diversity of commercial and widespread products, including cosmetics, food, rubber, paints and plastics, over the last decade. However, in spite of increasing in the population exposure to ZnO, the potential of genotoxicity induced by ZnO has not yet been clarified. To date, there are numerous studies associated with genotoxicity of ZnO, however, the overall verdict is still controversial. The biological effects of nanoparticles can be altered by their physicochemical properties, and within the laboratory, may be altered by the evaluation method used. Thus well-characterized chemicals and appropriate standardized methods are necessarily required for assessing genotoxicity of nanoparticles. In this study, we evaluated the genotoxicity of ZnO using four kinds of well-characterized ZnO nanoparticles: 20 nm negative charge, 20 nm positive charge, 70 nm negative charge, 70 nm positive charge. In vivo alkaline comet assay was conducted following guidelines approved by an international validation study group. Our results showed no statistically significant differences in the toxicity of in vivo comet assay of the four kinds of ZnO nanoparticles, compared to that of the solvent control. The present results suggest that ZnO nanoparticles may not induce genotoxicity, particularly in DNA strand breaks. Furthermore, these results have been generated in accordance with test guidelines, and hence, are considered as reliable information regarding ZnO induced genotoxicity.
Excessive reactive oxygen species (ROS) generation is one of the toxic mechanisms induced by gamma-ray irradiation. Redox factor is a critical player for controlling ROS levels in biosystems, and regulation of this factor by redox stimulators is of critical interest to a great number of areas of medical science. Selenium is well known to have a role in redox regulation and antioxidant function. One of the organic forms of selenium, selenomethionine (SeMet), is relatively nontoxic and can be applied orally to human. In this study, we investigated whether SeMet has a radioprotective effect or not in an animal model. To demonstrate this hypothesis, we conducted in vivo alkaline comet assay and histopathological analysis to determine whether SeMet exerts a radioprotective effect in animal models or not. Furthermore, gene expression profiles and signaling networks were analyzed using gene expression microarray. Also, quantitative real-time PCR were subsequently conducted to validate target genes. We found that gamma-ray-induced DNA damage in mouse spleen with SeMet pretreatment was significantly reduced in comparison to the one treated with gamma-ray alone. Using histopathological analysis, it showed that erythrocyte-containing red pulp was restored in mouse spleen pretreated with SeMet prior to gamma-ray exposure, whereas the red pulp was obviously eliminated in the mouse spleen treated with gamma-ray exposure alone. These results indicate the radioprotective effect of SeMet in mouse tissue, particularly spleen, toward gamma-ray-mediated DNA damage and cell abnormality. We also suggested potential target genes and signaling networks associated with the SeMet-mediated radioprotection mechanism. Our results would provide a critical scientific evidence for the further development of potential radioprotective agents. Furthermore, this information would be advantageous for radiation-related safety management as well as radiotherapeutics in public health fields.
The comet assay can be used to investigate the genotoxicity of various agents, including industrial chemicals, biocides, agrochemicals and pharmaceuticals. It has a number of advantages, such as: sensitivity for detecting low levels of DNA damages, the requirement for small numbers of cells per samples, low costs and ease of application. Nowadays, in vivo comet assay has been suggested as a potential tool for replacement of the in vivo rodent hepatocyte unscheduled DNA synthesis (UDS). International expert groups for comet assay have published recommendations describing standards which are aimed at establishing high quality protocols in order to obtain valid and reliable data. Despite that, international activity for validation of the in vivo comet assay has been carried out, and the distribution of protocol and interlaboratory validation have not yet been conducted in the Republic of Korea. Here, three laboratories participated in an inter-laboratory assessment to test the validity of the in vivo comet assay under the GLP (Good Laboratory Practice) system. Two genotoxic chemicals, N-methyl-N-nitrosourea (MNU) and ethyl methanesulfonate (EMS) were applied, as known and unknown chemicals, respectively, which were selected on the basis of the 3rd international validation study result. Among all GLP laboratories, the results showed a significant increase in DNA damage as % tail DNA as dose-related manner in both liver and stomach tissues treated with MNU relative to the negative control group. Similarly, the EMS-treated group also showed significantly increased % tail DNA damage, in a dose-dependent manner compared with the negative control group in both tissues. The in vivo comet results from all GLP laboratories in Republic of Korea were obviously consistent with the 3rd international validation study. In conclusion, we confirmed the interlaboratory validity of the in vivo comet assay using two genotoxic chemicals, which was assessed by three GLP laboratories in the Republic of Korea. This study can strengthen the in vivo comet assay to be suggested as an international standard protocol or guideline for genotoxicity testing of various genotoxic agents.