Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves 162-186).

Cancer is a major cause of morbidity and mortality affecting populations in all countries and all regions. Breast cancer (BC) is the second leading cause of cancer death among women in the UK. Although the overall survival of BC has been significantly improved due to systemic therapy in early BC, the treatment of advanced/metastatic BC remains a major challenge. The main limitation of therapeutic failure is the de novo and acquired resistance of BC cells to conventional anticancer drugs. Cancer stem cells (CSCs) have been thought to be responsible for the chemoresistance. My study demonstrated that mammospheres manifested CSC characteristics and are highly resistant to several first line anti-BC drugs. This may be due to the hypoxia in the centre of the spheres. Transfection of BC cells with NFκB p65 induces CSC characters and chemoresistance. Therefore hypoxia-induced activation of NFκB could lead to escape of CSCs from apoptosis and regenerate the tumour after conventional chemotherapy. In clinic, the relapsed cancer is commonly pan-resistant to various drugs. Development of CSCs-targeting drug will be significantly important in clinic for cancer patients. Disulfiram (DS) is a commercially available anti-alcoholism drug with strong cytotoxicity in a wide range of cancer types and has a reversing II effect on chemoresistance. In this study, the anticancer efficacy of DS on cancer cell lines and CSCs was investigated. DS was highly cytotoxic to BC cell lines in vitro in a copper (Cu)-dependent manner. CI-isobologram analysis demonstrated a synergistic effect between DS/Cu and paclitaxel (PAC) in BC cell lines. DS/Cu induces reactive oxygen species (ROS), activates JNK and p38 pathways and simultaneously inhibits NFκB activity in BC cell lines. DS/Cu may trigger intrinsic apoptotic pathway via modulation of the Bcl2 family. The in vitro clonogenicity and sphere-forming ability of BC cell lines were inhibited by DS/Cu. The common stem cell markers such as aldehyde dehydrogenase (ALDH) and CD24-/CD44+ as well as Nanog, Sox2, and Oct4 were also suppressed. In PAC resistant cell line, DS abolished CSC characters and completely reversed PAC resistance. Lipo-DS blocked NFκB activation and specifically targeted CSCs in vitro. Lipo-DS also targeted CSC population in vivo and showed very strong anticancer efficacy. This study elucidated the role of NFB in bridging hypoxia with CSC-related chemoresistance. It also investigated the fundamental anticancer mechanisms of DS. The results derived from this study indicate that further study may be able to translate DS into cancer therapeutics in the future.

Platinum-based chemotherapies are effective agents in the treatment of a wide variety of human cancers. However, patients with recurrent disease can become resistant to platinum-based chemotherapy, leading to low overall survival rates. Activating transcription factor 3 (ATF3) is a stress-inducible gene that is a regulator of cisplatin-induced cytotoxicity. ATF3 protein expression was upregulated after cytotoxic doses of cisplatin treatment in a panel of cell lines. A chromatin immunoprecipitation assay showed that upon treatment with cisplatin, ATF3 directly bound to the CHOP gene promoter and this correlated with an increase in CHOP protein expression. In a 1200 compound library screen performed on cancer cell lines, disulfiram, a dithiocarbamate drug, was identified as an enhancer of the cytotoxic effects of cisplatin. This increased cytotoxic action was likely due to disulfiram and cisplatin’s ability to induce ATF3 independently through two separate mechanisms, namely the MAPK and integrated stress pathways. Furthermore, ATF3 protein and mRNA levels were variable amongst human ovarian and lung cancer tissues, suggesting the potential for basal expression of ATF3 to be predictive of cisplatin treatment response. Thus, understanding ATF3’s role in cisplatin-induced cytotoxicity will lead to novel therapeutic approaches that could improve this drug’s efficacy.

Platinum-based chemotherapies are effective agents in the treatment of a wide variety of human cancers. However, patients with recurrent disease can become resistant to platinum-based chemotherapy, leading to low overall survival rates. Activating transcription factor 3 (ATF3) is a stress-inducible gene that is a regulator of cisplatin-induced cytotoxicity. ATF3 protein expression was upregulated after cytotoxic doses of cisplatin treatment in a panel of cell lines. A chromatin immunoprecipitation assay showed that upon treatment with cisplatin, ATF3 directly bound to the CHOP gene promoter and this correlated with an increase in CHOP protein expression. In a 1200 compound library screen performed on cancer cell lines, disulfiram, a dithiocarbamate drug, was identified as an enhancer of the cytotoxic effects of cisplatin. This increased cytotoxic action was likely due to disulfiram and cisplatin’s ability to induce ATF3 independently through two separate mechanisms, namely the MAPK and integrated stress pathways. Furthermore, ATF3 protein and mRNA levels were variable amongst human ovarian and lung cancer tissues, suggesting the potential for basal expression of ATF3 to be predictive of cisplatin treatment response. Thus, understanding ATF3’s role in cisplatin-induced cytotoxicity will lead to novel therapeutic approaches that could improve this drug’s efficacy.

Platinum-based chemotherapies are effective agents in the treatment of a wide variety of human cancers. However, patients with recurrent disease can become resistant to platinum-based chemotherapy, leading to low overall survival rates. Activating transcription factor 3 (ATF3) is a stress-inducible gene that is a regulator of cisplatin-induced cytotoxicity. ATF3 protein expression was upregulated after cytotoxic doses of cisplatin treatment in a panel of cell lines. A chromatin immunoprecipitation assay showed that upon treatment with cisplatin, ATF3 directly bound to the CHOP gene promoter and this correlated with an increase in CHOP protein expression. In a 1200 compound library screen performed on cancer cell lines, disulfiram, a dithiocarbamate drug, was identified as an enhancer of the cytotoxic effects of cisplatin. This increased cytotoxic action was likely due to disulfiram and cisplatin’s ability to induce ATF3 independently through two separate mechanisms, namely the MAPK and integrated stress pathways. Furthermore, ATF3 protein and mRNA levels were variable amongst human ovarian and lung cancer tissues, suggesting the potential for basal expression of ATF3 to be predictive of cisplatin treatment response. Thus, understanding ATF3’s role in cisplatin-induced cytotoxicity will lead to novel therapeutic approaches that could improve this drug’s efficacy.

Cancer, a global epidemic, is a major cause of morbidity and mortality affecting populations in all nations and regions. Breast cancer (BC) is the second most common cancer in the world and the most fatal malignancy affecting women both in the developed and developing countries. Even with the improvement in overall survival of BC patients due to early detection and advancement with systematic therapy, triple negative breast cancer (TNBC), an aggressive subtype of BC still remains a major challenge as it lacks targetable receptors. Chemotherapy is the main treatment for TNBC. However, de novo and acquired resistance to conventional anticancer drugs is a major limitation and cause of therapeutic failure. Cancer stem cells (CSCs) are believed to be responsible for chemoresistance and tumour relapse. My study demonstrates that hypoxia is involved in the development and maintenance of these CSCs traits in TNBC, as cells grown in hypoxia are significantly resistant to several first line anti-BC drugs. Hypoxia-induced activation of nuclear factor kappa B (NFB) and hypoxia inducible factors (HIFs) also play pivotal roles in chemoresistance. Forced expression of NFB and HIFs by transfection with p65 subunits of NFB and HIF1α and 2α subunits induced CSCs characters and resistance to a range of anticancer drugs in TNBC cell lines. My study also indicated a positive loop between the activation of NFB and HIFs. Therefore development of novel medicine to interfere the pathways of hypoxia and NFB may efficaciously target CSCs and reverse chemoresistance which will be of clinical significance for TNBC treatment. iv Disulfiram (DS) is a commercially available anti-alcoholism drug. Recent studies demonstrate that it is highly cytotoxic in a wide range of cancer types and potentially repurposed as an anticancer drug. The anticancer mechanisms of DS were investigated in this study. The results from my study indicate that the cytotoxicity of DS is copper (Cu) dependent with a biphasic manner. The instant cytotoxic phase is induced by the extracellular reactive oxygen species (ROS) generated by the reaction between DS and Cu. The delayed killing is caused by the complex diethyldithiocarbamate (DDC) and Cu (DDC-Cu), the final product of the reaction. The cytotoxicity of both phases needs the intact DS. However, due to the extremely short half-life of DS in the bloodstream, the anticancer efficacy of DS has been severely hampered in vivo and in patients. Nanotechnology-based drug delivery system is a rapidly evolving and expanding interdisciplinary field involving in an amalgamation of chemistry, engineering, biology and medicine. In the last part of my study, I have successfully encapsulated DS into polymeric micelle (PM) nanoparticles. The half-life of PM encapsulated DS (PM-DS) was extended to over 3 hours in horse serum. The PM-DS showed strong anticancer efficacy. Therefore this nano-enabled DS may be able to translate DS into cancer therapeutics in the future.

Platinum-based chemotherapies are effective agents in the treatment of a wide variety of human cancers. However, patients with recurrent disease can become resistant to platinum-based chemotherapy, leading to low overall survival rates. Activating transcription factor 3 (ATF3) is a stress-inducible gene that is a regulator of cisplatin-induced cytotoxicity. ATF3 protein expression was upregulated after cytotoxic doses of cisplatin treatment in a panel of cell lines. A chromatin immunoprecipitation assay showed that upon treatment with cisplatin, ATF3 directly bound to the CHOP gene promoter and this correlated with an increase in CHOP protein expression. In a 1200 compound library screen performed on cancer cell lines, disulfiram, a dithiocarbamate drug, was identified as an enhancer of the cytotoxic effects of cisplatin. This increased cytotoxic action was likely due to disulfiram and cisplatin’s ability to induce ATF3 independently through two separate mechanisms, namely the MAPK and integrated stress pathways. Furthermore, ATF3 protein and mRNA levels were variable amongst human ovarian and lung cancer tissues, suggesting the potential for basal expression of ATF3 to be predictive of cisplatin treatment response. Thus, understanding ATF3’s role in cisplatin-induced cytotoxicity will lead to novel therapeutic approaches that could improve this drug’s efficacy.

This study was designed to determine whether the Minnesota Multiphasic Personality Inventory could successfully predict the outcome of rehabilitative treatment given 84 racially mixed male alcoholics having a mean age of 42.2 years who were admitted to the Wyoming State Hospital between September 1, 1965, and September 1, 1966. The subjects were given the MMPI before receiving 16 weeks of treatment consisting of education, group and individual psychotherapy, alcoholics anonymous, routine "ward treatment," disulfiram (antibuse), special ward unit, and special program without ward. After their release from hospital, the subjects were evaluated yearly for five years by relatives, employers, and themselves concerning vocational and familial responsibilities, sobriety, AA attendance, and drinking. Multiple regression analysis, a multivariate statistical technique was utilized to analyze the data from a systematic follow-up questionnaire. At the end of five years 32 of the original 84 subjects could be located. The study concluded that the MMPI cannot be used to predict successful treatment of alcoholics at Wyoming State Hospital.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23565号 / 医博第4779号 / 新制||医||1054(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 万代 昌紀, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Triple negative breast cancer (TNBC) represents an aggressive subtype for which only chemotherapy is available. The RB1 tumour suppressor is frequently lost in human breast cancer, primarily in TNBC. Loss of RB1 deregulates the cell cycle and is thought to affect BC response to endocrine, radiation, and chemotherapy. However, the global chemosensitivity of Rb null BC is not known. Here I demonstrate that RB1-deficient TNBC cells are highly sensitive to radiation, and moderately sensitive to doxorubicin and methotrexate. However, loss of RB1 does not increase sensitivity to multiple other drugs. Moreover, a non-biased screen of 2 RB-deficient versus 2 RB-proficient lines with ~3500 drugs did not reveal any difference in sensitivity, but identified disulfiram as a potent drug, which compared favourably with current chemotherapeutics against TNBC. Disulfiram’s efficacy was validated against 13 human TNBC lines with an average IC50 of 300nM. IQGAP1 was identified as a potential target of disulfiram.