Wang Huan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 148-158). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.I / LIST OF FIGURES AND TABLES --- p.VIII / ABSTRACT --- p.1 / 摘要 --- p.3 / Chapter CHAPTER 1 --- GENERAL INTRODUCTION / Chapter I. --- The essential factors related to cancer --- p.5 / Chapter a. --- Carcinogens --- p.5 / Chapter b. --- Carcinogenesis pathways --- p.7 / Chapter c. --- DNA adducts formation and breast cancer --- p.7 / Chapter II. --- Cytochrome P450 I enzyme family --- p.8 / Chapter a. --- CYP450 superfamily --- p.8 / Chapter b. --- CYP1A1 --- p.10 / Chapter c. --- CYP1B1 --- p.11 / Chapter III. --- Transactivation of CYP1 enzymes by aryl hydrocarbon receptor (AhR) --- p.12 / Chapter IV. --- Phase II enzyme UGT and cancer prevention --- p.13 / Chapter V. --- Estrogen metabolism and the hormone-dependent breast cancer --- p.15 / Chapter a. --- Estrogen and breast cancer initiation --- p.15 / Chapter b. --- Estrogen Receptor (ER) --- p.15 / Chapter c. --- Estradiol hydroxylation pathways --- p.15 / Chapter VI. --- Phytochemicals and cancer prevention --- p.18 / Chapter VII. --- Outline of this study --- p.20 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter I. --- Chemicals --- p.21 / Chapter II. --- Cell culture and treatments --- p.21 / Chapter 1. --- Maintenance of cells --- p.21 / Chapter 2. --- Preparation of cell stock --- p.22 / Chapter 3. --- Cell recovery from liquid nitrogen stock --- p.22 / Chapter 4. --- Measurement of cell viability --- p.22 / Chapter 5. --- Preparation of cell lysates --- p.23 / Chapter 6. --- XRE-luciferase gene reporter assay --- p.23 / Chapter a. --- Transient transfection of cell using lipofectamine PLUS reagent --- p.23 / Chapter b. --- Dual Luciferase Assay --- p.24 / Chapter III. --- Enzyme Activities --- p.24 / Chapter 1. --- Isolation of microsomes --- p.24 / Chapter 2. --- EROD activities in intact cells --- p.24 / Chapter 3. --- EROD inhibition assay --- p.25 / Chapter IV. --- Manipulation of Nuclear Acid --- p.26 / Chapter 1. --- Preparation of transfected DNA --- p.26 / Chapter a. --- Separation and purification of DNA from agarose gel --- p.26 / Chapter b. --- Restriction digestion --- p.26 / Chapter c. --- Ligation of DNA fragments --- p.27 / Chapter d. --- Transformation of DH5a --- p.27 / Chapter e. --- Small scale plasmid purification from DH5a (mini prep) --- p.28 / Chapter f. --- Large scale plasmid isolation from DH5a (maxi-prep) --- p.28 / Chapter g. --- Construction of XRE activated luciferase reporter gene --- p.29 / Chapter 2. --- Measurement of DMBA-DNA adduct formation --- p.29 / Chapter 3. --- Semi-quantitative RT-PCR Assay --- p.30 / Chapter a. --- Isolation of RNA using TRIzol® Reagent --- p.30 / Chapter b. --- RT-PCR --- p.31 / Chapter V. --- Phase II enzyme-UGT activity assay --- p.32 / Chapter VI. --- HPLC for estradiol-hydroxylation analysis --- p.33 / Chapter 1. --- HPLC condition for hydroxyestradiol separation and measurement --- p.33 / Chapter 2. --- Determination of microsomal estradiol hydroxylase activity --- p.34 / Chapter 3. --- Assay of estradiol metabolism in MCF-7 cells --- p.34 / Chapter VII. --- Statistical Analysis --- p.35 / Chapter CHAPTER 3 --- CHALCONES ANTAGONIZE DMBA-INDUCED CARCINOGENESIS BY MODULATION OF CYP1A1/1B1 AND UGT ACTIVITIES / Chapter Part One --- Introduction --- p.36 / Chapter Part Two --- Results --- p.40 / Chapter Section One --- Chalcones antagonize DMBA carcinogenesis by inhibiting CYP1A1 and CYP1B1 activities --- p.40 / Chapter I. --- Chalcones inhibited DMBA-induced EROD activities in MCF-7 cells --- p.40 / Chapter II. --- Inhibition of chalcones on microsomal CYP1A1 & 1B1 enzyme activities --- p.43 / Chapter III. --- Reduction of DMBA-induced DNA adduct by chalcones --- p.52 / Chapter IV. --- Chalcones antagonized CYP1A1 XRE transactivation --- p.54 / Chapter V. --- Chalcones suppressed DMBA-induced CYP1 gene expression --- p.56 / Chapter Section Two --- Chalcones modulate DMBA carcinogenesis by regulating UGT activities --- p.63 / Chapter I . --- Chalcones regulated UGT1A1 gene expression in MCF-7 cells --- p.63 / Chapter II. --- Chalcones affected UGT enzyme activity in HepG2 cells --- p.70 / Chapter III. --- Chalcones regulated UGT1A1 gene expression in HepG2 cells --- p.73 / Chapter Part Three --- Discussion --- p.80 / Chapter I . --- Chalcones are potential chemopreventive agents --- p.80 / Chapter II. --- Chalcones modulated Phase I enzyme activities --- p.80 / Chapter III. --- Chalcones regulated Phase II enzyme activities --- p.82 / Chapter IV. --- Chalcones suppressed DMBA-induced DNA-adduct formation in MCF-7 cells --- p.82 / Chapter V. --- The anti-carcinogenic properties of chalcones and their structures --- p.83 / Chapter CHAPTER 4 --- EFFECTS OF PERILLYL ALCOHOL AND LIMONENE ON CYP1 AND UGT ENZYMES / Chapter Part One --- Introduction --- p.85 / Chapter Part Two --- Results --- p.87 / Chapter I. --- Perillyl alcohol and limonene modulated DMBA-induced CYP1A1/1B1 activities in MCF-7 cells --- p.87 / Chapter II. --- Perillyl alcohol and limonene regulated microsomal CYP1A1/1B1 activities --- p.89 / Chapter III. --- Perillyl alcohol and limonene regulated DMBA-induced DNA adduct formation in MCF-7 cells --- p.93 / Chapter IV. --- Perillyl alcohol and limonene regulated CYP1A1 & CYP1B1 gene expressions in MCF-7 cells --- p.95 / Chapter V. --- Effect of perillyl alcohol on CYP1A1 XRE transactivation --- p.97 / Chapter VI. --- Cytotoxic effect of perillyl alcohol and limonene on MCF-7 cells --- p.98 / Chapter VII. --- Perillyl alcohol and limonene modulated UGT1A1 gene expression in MCF-7 cells --- p.99 / Chapter VIII. --- Perillyl alcohol and limonene modulated UGT enzyme in HepG2 cells --- p.101 / Chapter Part Three --- Discussion --- p.106 / Chapter CHAPTER 5 --- LYCOPENE MEDIATED DMBA-INDUCED PHASE I & PHASE II ENZYME ACTIVITIES AND GENE EXPRESSIONS / Chapter Part Three --- Introduction --- p.109 / Chapter I. --- Biochemical properties of lycopene --- p.109 / Chapter II. --- Bioavailability of lycopene --- p.110 / Chapter III. --- Lycopene and cancers in hormonal sensitive tissues --- p.110 / Chapter Part Two --- Results --- p.111 / Chapter I . --- Lycopene modulated DMBA-induced CYP1A1/1B1 activities in MCF-7 cells --- p.111 / Chapter II. --- Lycopene competitively inhibited microsomal CYP1A1 & CYP1B1 activities --- p.113 / Chapter III. --- Lycopene suppressed DMBA-induced DNA adduct formation in MCF-7 cells --- p.115 / Chapter IV. --- Lycopene regulated CYP1A1 & CYP1B1 gene expression in MCF-7 cells --- p.116 / Chapter V. --- Effect of lycopene on CYP1A1 XRE trasactivation --- p.117 / Chapter VI. --- Cytotoxic effect of lycopene on MCF-7 cells --- p.118 / Chapter VII. --- Lycopene modulated UGT enzyme in MCF-7 cells --- p.119 / Chapter VIII. --- Lycopene modulated UGT enzyme in HepG2 cells --- p.121 / Chapter Part Three --- Discussion --- p.123 / Chapter CHAPTER 6 --- CHALCONES AND PERILLYL ALCOHOL REGULATEDCYP1A1 & CYP1B1 MEDIATED ESTRADIOL METABOLIZING PATHWAYS / Chapter Part One --- Introduction --- p.125 / Chapter I . --- Estrogen hydroxylation and human breast cancer risk --- p.125 / Chapter II. --- CYP1 enzymes catalyze estradiol-hydroxylation in human breast cancer cells --- p.126 / Chapter III. --- Phytochemicals mediate estrogen-hydroxylation pathways --- p.126 / Chapter Part Two --- Estrogen metabolite detection and separation by HPLC --- p.127 / Chapter Part Three --- Results --- p.129 / Chapter I . --- Perillyl alcohol modulated CYP1A1 & CYP1B1-mediated Estradiol hydroxylation --- p.129 / Chapter II. --- Kinetics assays of chalcones on CYP1A1 & CYP1B1 microsomes induced estradiol hydroxylation --- p.131 / Chapter III. --- Chalcones suppressed Estradiol-hydroxylase activities in MCF-7 cells --- p.137 / Chapter Part Four --- Discussion --- p.140 / Chapter CHAPTER 7 --- SUMMARY / Chapter I . --- Chalcones displayed inhibitory effects on DMBA-induced carcinogenesis --- p.142 / Chapter II. --- Perillyl alcohol and limonene modulated DMBA-induced carcinogenesis --- p.143 / Chapter III. --- Lycopene also possessed chemoproventive properties --- p.143 / APPENDIX 1 ABBREVIATIONS --- p.144 / APPENDIX 2 REAGENTS --- p.145 / APPENDIX 3 PRIMER LISTS --- p.147 / REFERENCE --- p.148

Considerada como uma nova estratégia no direcionamento de fármacos para tecidos lesionados, a LDE é uma nanopartícula lipídica que se concentra em locais inflamatórios com altas taxas de proliferação celular, como lesões ateroscleróticas, e é usada em modelos experimentais e no homem. Docetaxel (DTX), agente quimioterápico antiproliferativo, ainda não foi explorado no tratamento da aterosclerose. Coelhos New Zealand brancos machos foram alimentados com ração enriquecida com 1% de colesterol para induzir aterosclerose ao longo do período experimental de 8 semanas. Após 4 semanas, os animais foram tratados semanalmente com LDE-DTX (n =9) com dose de 1 mg/kg e.v. ou apenas com LDE (grupo Controle, n =9). O consumo de ração e os perfis lipídico, hematológico e ponderal foram avaliados durante o protocolo nos tempos basal, 4 semanas e final. Após a eutanásia, foram realizadas análises morfológicas e Western blot das aortas. Como esperado, o colesterol total aumentou aproximadamente 42 vezes em ambos os grupos quando comparados os períodos basal e final. Houve diminuição no número de hemácias entre os períodos basal e final em ambos os grupos, aparentemente não relacionada ao tratamento. Os animais não apresentaram toxicidade renal e hepática. A área de lesão macroscópica nas aortas do grupo LDE-DTX foi aproximadamente 80% menor em relação ao Controle e a área de placa na região do arco aórtico foi 86% menor no grupo tratado com LDE-DTX quando comparado ao Controle. Em relação aos fatores inflamatórios, a expressão proteica de CD68 foi 64% menor no grupo tratado com LDE-DTX quando comparado ao grupo Controle. MCP-1 foi 84% menor no grupo tratado com LDEDTX e o TNF-alfa foi 44% menor no grupo tratado, quando comparados ao grupo Controle. A expressão proteica de interleucina IL-1beta e do NFkB foram cerca de 60% menores no grupo tratado assim como a IL-6, que foi 79% menor, ambos comparados ao grupo Controle. O fator de von Willebrand foi cerca de 30% menor no grupo tratado com LDE-DTX comparado ao Controle. Os fatores próapoptóticos apresentaram menor expressão no grupo LDE-DTX: a caspase 3 foi 82% menor em comparação ao Controle, caspase 9 e Bax, cerca de 50% menor que o controle bem como o fator anti-apoptótico Bcl-2. A expressão proteica dos colágeno I e III também foi menor no grupo LDE-DTX. Comparados ao controle, a expressão proteica de MMP-2 e MMP-9 foram cerca de 70% menores no grupo LDE-DTX. O marcador de proliferação celular PCNA foi 41% menor no grupo LDE-DTX em comparação com o grupo Controle. O tratamento com a associação LDE-DTX mostrou-se eficaz, uma vez que os coelhos tratados apresentaram uma área menor da lesão aterosclerótica, menor inflamação, morte celular e proliferação na aorta quando comparado ao grupo Controle / Considered as a new strategy of targeting drugs to injured tissues, LDE, a lipid core nanoparticle concentrates on inflammatory sites with high cell proliferation rates, as atherosclerotic lesions, and it is used as a vehicle for drugs in experimental models and in humans. Docetaxel (DTX), an antiproliferative chemotherapeutic agent has not been explored yet in the treatment of atherosclerosis. New Zealand white male rabbits were fed with 1% cholesterol diet throughout the 8-week experimental period to induce atherosclerosis. After 4 weeks, the animals were treated weekly with LDE-DTX (n=9) at a dose of 1mg/kg i.p. or only with LDE (Control group, n=9). We evaluated feed intake, lipid, hematological, and weight profiles during the protocol at baseline, 4 weeks and post-treatment. After euthanasia was performed morphological analysis and Western blot of the aorta. As expected, total cholesterol increased 42-fold in both groups comparing baseline to post-treatment. There was a decrease in red blood cells number in both groups but it is probably not treatment-related. There was no hepatic and renal treatment-related toxicity. The macroscopic lesion area in the aortas of LDE-DTX was approximately 80% smaller compared to Control and the morphometry of the aortic arch was 86% smaller in LDE-DTX group compared to Control group. Regarding inflammatory factors, CD68 was 64% lower in LDEDTX group comparing to Control group. MCP-1 was 84% in LDE-DTX group and TNF-alpha was 44% lower in the treated group comparing to Control group. The protein expression of IL-1beta and NFkB were about 60% lower in the LDE-DTX group as well as IL-6 that was 79% lower, both compared to Control group. The von Willebrand factor was about 30% lower in LDE-DTX group compared to Control group. The pro-apoptotic factors showed lower expression in LDE-DTX group: caspases 3 was 82% lower compared to control and caspases 9 and Bax were about 50% than Control group as well as the anti-apoptotic factor Bcl-2. The protein expression of collagen I and III were lower in LDE-DTX group. Compared to control, the protein expression of MMP-2 and MMP-9 were about 70% lower in the LDE-DTX group compared to Control group. The cell proliferation marker PCNA was 41% lower in LDE-DTX group compared to Control group. Treatment with LDE-DTX association proved to be effective since the treated rabbits had a smaller area of the atherosclerotic lesion, lower inflammation, cell death and proliferation in the aorta when compared to Control group

Cheung, Ka Yan. / "Aug 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 157-167). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgments --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xi / Abbreviations --- p.xii / Chapter Chapter1 --- General Introduction --- p.1 / Chapter 1.1 --- Cancer epidemiology and managements --- p.2 / Chapter 1.2 --- Photodynamic therapy (PDT) as cancer treatment --- p.7 / Chapter 1.3 --- Pheophorbide a (Pa) as a photosensitizer for PDT --- p.13 / Chapter 1.4 --- Aim of study --- p.15 / Chapter Chapter2 --- The anti-proliferative effect of pheophorbide a- mediated photodynamic therapy on human breast adenocarcinoma cell line MCF-7 --- p.17 / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- Cell cycle regulation --- p.18 / Chapter 2.1.2 --- Growth arrest and DNA damage inducible (GADD) genes as cell cycle regulators --- p.22 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Materials / Chapter 2.2.1.1 --- Cell line --- p.29 / Chapter 2.2.1.2 --- "Cell culture medium, supplements and other reagents" --- p.29 / Chapter 2.2.1.3 --- Gene expression assay reagents --- p.30 / Chapter 2.2.1.4 --- Reagents and buffers for Western blotting --- p.32 / Chapter 2.2.1.5 --- Cell cycle analysis reagents --- p.35 / Chapter 2.2.2 --- Methods / Chapter 2.2.2.1 --- Cell line propagation and subculture --- p.36 / Chapter 2.2.2.2 --- Whole-transcript expression micro array analysis --- p.37 / Chapter 2.2.2.3 --- GADD genes expression assay- RT-PCR --- p.37 / Chapter 2.2.2.4 --- Cell cycle analysis --- p.40 / Chapter 2.2.2.5 --- Western Blotting --- p.41 / Chapter 2.2.2.6 --- Statistical analysis --- p.43 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Effect of Pa-PDT on GADD genes expression by whole-transcript expression microarray analysis --- p.44 / Chapter 2.3.2 --- Effect of Pa-PDT on GADD genes expression by RT-PCR --- p.46 / Chapter 2.3.3 --- Temporal change in the cell cycle profile after Pa-PDT --- p.48 / Chapter 2.3.4 --- Effect of Pa-PDT on cell cycle associated proteins --- p.65 / Chapter 2.4 --- Discussion --- p.67 / Chapter Chapter3 --- Development of drug resistance in human breast adenocarcinoma cell line MDA and the circumvention by pheophorbide a-mediated photodynamic therapy --- p.77 / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Clinical Importance of multidrug resistance (MDR) --- p.78 / Chapter 3.1.2 --- Mechanisms of MDR --- p.78 / Chapter 3.1.3 --- Development of MDR cell lines --- p.82 / Chapter 3.1.4 --- Reversal of MDR by P-glycoprotein modulators --- p.83 / Chapter 3.1.5 --- Therapeutic potential of Pa-PDT in treating MDR cancers --- p.83 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Materials / Chapter 3.2.1.1 --- Cell line --- p.85 / Chapter 3.2.1.2 --- "Cell culture medium, supplements and other reagents" --- p.85 / Chapter 3.2.1.3 --- Cell viability assay reagents --- p.85 / Chapter 3.2.1.4 --- Gene expression assay reagents --- p.86 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Cell line propagation and subculture --- p.87 / Chapter 3.2.2.2 --- Drug-resistance development --- p.88 / Chapter 3.2.2.3 --- Measurement of cell viability - MTT reduction assay --- p.88 / Chapter 3.2.2.4 --- ABCB1 expression assay- RT-PCR --- p.89 / Chapter 3.2.2.5 --- Doxorubicin uptake assay --- p.91 / Chapter 3.2.2.6 --- Pheophorbide a uptake assay --- p.91 / Chapter 3.2.2.7 --- Statistical analysis --- p.92 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Cytotoxicity of doxorubicin on MDA and MDA-R cells --- p.93 / Chapter 3.3.2 --- mRNA expression of ABCB1 (P-glycoprotein) in MDA and MDA-R cells --- p.96 / Chapter 3.3.3 --- Doxorubicin uptake by MDA and MDA-R cells --- p.98 / Chapter 3.3.4 --- Circumvention of drug resistance in MDA-R cells by Pa-PDT --- p.102 / Chapter 3.3.5 --- Pheophorbide a uptake by MDA and MDA-R cells --- p.104 / Chapter 3.4 --- Discussion --- p.106 / Chapter Chapter4 --- Synergistic anti-proliferation of pheophorbide a-mediated photodynamic therapy and doxorubicin on multidrug resistant uterine sarcoma cell line Dx5 --- p.113 / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- Clinical limitations of doxorubicin as chemotherapeutic drug --- p.114 / Chapter 4.1.2 --- Clinical limitations of photodynamic therapy --- p.115 / Chapter 4.1.3 --- Combination therapy with Dox and Pa-PDT --- p.117 / Chapter 4.1.4 --- Uterine sarcoma cell line Dx5 as in vitro model for combination therapy --- p.118 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1 --- Materials / Chapter 4.2.1.1 --- Cell line --- p.120 / Chapter 4.2.1.2 --- "Cell culture medium, supplements and other reagents" --- p.120 / Chapter 4.2.1.3 --- Anti-cancer drugs --- p.121 / Chapter 4.2.1.4 --- "ROS inhibitor, α-tocopherol" --- p.121 / Chapter 4.2.1.5 --- Cell viability assay reagents --- p.122 / Chapter 4.2.1.6 --- P-glycoprotein activity assay reagents --- p.122 / Chapter 4.2.2 --- Methods - / Chapter 4.2.2.1 --- Cell line propagation and subculture --- p.123 / Chapter 4.2.2.2 --- Cell viability assay --- p.123 / Chapter 4.2.2.3 --- P-glycoprotein activity assay --- p.124 / Chapter 4.2.2.4 --- Statistical analysis --- p.125 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Combination therapy of Pa-PDT and doxorubicin in Dx5 cells --- p.126 / Chapter 4.3.2 --- Effect of α-tocopherol on the synergism between Pa-PDT and doxorubicin in Dx5 cells --- p.129 / Chapter 4.3.3 --- Effect of Pa-PDT on P-glycoprotein activity in Dx5 cells --- p.132 / Chapter 4.3.4 --- Combination therapy of Pa-PDT and doxorubicin in SA cells --- p.138 / Chapter 4.4 --- Discussion --- p.141 / Chapter Chapter5 --- General Discussion --- p.148 / Chapter 5.1 --- Pa-PDT induced growth arrest and DNA fragmentation in breast cancer MCF-7 cells --- p.149 / Chapter 5.2 --- Circumvention of doxorubicin resistance by Pa-PDT in breast cancer MDA cells --- p.151 / Chapter 5.3 --- Synergistic anti-proliferation of Pa-PDT and doxorubicin on uterine sarcoma cell line Dx5 --- p.151 / Chapter 5.4 --- Clinical implication --- p.153 / Chapter 5.5 --- Conclusions and future perspectives --- p.153 / References --- p.157

OBJETIVO: Avaliar a eficácia e segurança da imunoterapia tópica com imiquimode creme 5% no tratamento do carcinoma basocelular nodular periocular. MÉTODOS: Pacientes com carcinoma basocelular confirmado por biopsia e com contraindicação clínica para a cirurgia reconstrutiva devido ao alto risco ou que recusaram a cirurgia por razões estéticas ou fobia foram incluídos no estudo. O tratamento foi iniciado após treinamento do paciente e de acompanhante. A posologia foi de 5 vezes por semana por 8 a 16 semanas. Acompanhamento quinzenal foi realizado durante a vigência do tratamento com questionário, exame biomicroscópico, medida da acuidade visual e documentação fotográfica. As características clínicas das lesões foram mensuradas através do software ImageJ. Após 12 semanas do fim da terapia, uma nova biópsia na região da lesão foi guiada por fotografia. O seguimento dos pacientes foi semestral, após fim do tratamento, com biópsias anuais da região até o presente momento. RESULTADOS: 19 foram tratadas. A taxa de cura histológica foi de 89,5% após três meses do final do tratamento, e de 84,2% nos três anos de seguimento (39,5 meses). A taxa de cura histológica em três anos foi de 100% para lesões menores que 10 mm, e de 81,8% para lesões maiores que 10 mm. De uma forma geral, os efeitos colaterais da medicação foram mais frequentes durante as oito primeiras semanas de tratamento. Quanto menor foi a distância da lesão à margem palpebral, maior foi chance de o paciente desenvolver ectrópio no tratamento (p = 0,045). Assim, como quanto maior foi a inflamação, maior foi a chance de desenvolver ectrópio, dor e edema durante o tratamento (p = 0,017, p = 0,016 e p = 0,044, respectivamente). CONCLUSÕES: Imiquimode creme 5% mostrou-se eficaz para o tratamento alternativo do carcinoma basocelular periocular, principalmente em lesões menores que 10 mm. Em adição, demonstrou um interessante efeito neoadjuvante sobre as lesões maiores que 10 mm que não foram curadas. Mostrou-se um tratamento seguro; entretanto, um cuidado maior deve ser dado às lesões próximas à margem palpebral devido ao maior risco de complicações e desenvolvimento de ectrópio / Objective: to evaluate the efficacy and safety of topical administration of 5% imiquimod cream in the treatment of periocular nodular basal cell carcinoma (BCC). Methods: Patients with periocular nodular basal cell carcinoma confirmed by biopsy and clinical contraindication to reconstructive surgery due to high risk or who decline surgery for aesthetic reasons or phobia were included in the study. The medication was applied once a day, five days a week for 8-16 weeks. Treatment was initiated after provision of patient and caretaker training. During treatment, patients were followed up twice a month with questionnaires, biomicroscopic examinations, measurement of visual acuity and photographic documentation. The clinical characteristics of the tumors were registered with the software ImageJ. Twelve weeks after the end of treatment, an image-guided biopsy of the tumor site was performed. Patients have since been attending follow-up visits every six months, and biopsies of the region are performed annually. Results: 19 tumors were treated with imiquimod. The average histological cure rate was 89.5% after 3 months at the end of the treatment and 84.2% after 3 years of follow-up (39.5 months). The 3-year histological cure rate was 100% for smaller tumors and 81.8% for larger tumors (>10 mm). In general, drug-related side effects were more frequent during the first 8 weeks of treatment. The smaller the distance between tumor and lid margin, the greater the probability of developing ectropion during treatment (p=0.045). Likewise, the more severe the inflammation, the greater the probability of developing ectropion, pain and edema during treatment (p=0.017, p=0.016 and p=0.044 respectively). Conclusion: Topical administration of 5% imiquimod cream was found to be an efficacious and relatively safe alternative treatment for periocular BCC, especially for tumors smaller than 10 mm, with interesting neoadjuvant effects on uncured tumors larger than 10 mm. However, special care is required when treating tumors near the eyelid margin due to the risk of complications and development of ectropion

Thesis (Ph.D.)--University of Toledo, 2007. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: James Willey. Includes abstract. Title from title page of PDF document. Bibliography: pages 47-51, 73-76, 120-174.

The antimutagenic potency of wheat grain and berry extracts was studied in vitro against several heterocyclic amines (HCAs) using the Salmonella mutagenicity assay and the anticarcinogencity of wheat grain was studied in vivo using the rat colonic aberrant crypt focus assay. Wheat bran, which binds HCAs in vitro, as well as refined wheat and unrefined whole wheat, inhibited the mutagenic activities of 2-amino-3- methylimidazo [4, 5-f] quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) when they were co-incubated and the supernatant (minus grain) was added to the Salmonella mutagenicity assay. The water-soluble fraction alone from refined and unrefined wheat, but not bran, also inhibited these mutagens in vitro. In vivo, AIN- 93G diets containing refined wheat or unrefined wheat were examined for their ability to inhibit IQ-induced colonic aberrant crypt foci (ACF) in the F344 rat. A slight increase in the number of aberrant crypts/ACF (AC/ACF) was seen after 16 weeks in rats treated post-initiation with refined wheat (p<0.05), and fewer foci with 2 or 3 aberrant crypts (ACF-2) were found in rats given unrefined whole wheat post-initiation compared with animals treated with the same diet during the initiation phase (p<0.05). There was no significant difference in the profile of IQ urinary metabolites or excretion of promutagens 0-48 hours after carcinogen dosing, and grains had no effect on hepatic cytochrome P450 (CYP) 1A1, CYP1A2, aryl sulfotransferase, or N-acetyltransferase activities; however, a slightly higher UDP-glucuronosyl transferase activity was observed in rats fed unrefined wheat compared with refined wheat diets (p<0.05). Thus, despite their antimutagenic activities in vitro, only marginal effects were seen with refined and unrefined wheat in vivo with respect to induction of hepatic enzyme activities, carcinogen metabolism, or IQ-induced ACF in the rat colon. The fresh juice and extract of crandall black currant (Ribes aureum) were not mutagens in the Salmonella mutagenicity assay. Berry extract or fresh juice at levels to 50 ��l (22 mg berry) in a 500 ��l pre-incubation system significantly inhibited the mutagenicity of IQ, a mutagen from cooked meat, by 32% when rat liver S9 bioactivation system was present. One hundred ��l of crandall black currant extract gave 89% inhibition of IQ mutagenicity (p<0.05). However, the mutagenicity of 2-hydroxyamino-3-methylimidazo[4,5-f] quinoline (N-hydroxy- IQ), a direct-acting metabolite of IQ, was not affected. An in vitro fluorometric assay showed the activity of cytochrome P 450 (CYP) 1A1 and CYP 1A2 was decreased. Inhibition of CYP 1A2 activity may be an important mechanism of antimutagenicity of crandall black currant extract. Similar results were also observed with other berry samples. Key word: cereal grains, black currant, berry, aberrant crypt foci, heterocyclic amines, CYP1A1, CYP1A2, Salmonella mutagenicity assay. / Graduation date: 2003

N-(4-Hydroxyphenyl) retinamide (4-HPR) is a derivative of all-trans-retinoic acid that induces apoptosis in cancer cell lines and is being tested in clinical trials as a relatively non-toxic anti-cancer agent. 4-HPR induces de novo sphingolipid biosynthesis and production of ceramide has been suggested to contribute to the growth arrest and apoptosis. To characterize the types of ceramides that might be involved, we used liquid chromatography, electrospray ionization tandem mass spectrometry (LC ESI-MS/MS) to analyze the sphingolipids, and found that 4-HPR increased total sphingolipid amounts, but unexpectedly, ceramides (i.e., N-acylsphingosines) changed very little, and in some cases decreased. Instead, dihydroceramides (i.e., N-acylsphinganines) increased as much as 10-fold, both as the free species and as the backbones of dihydrosphingomyelins and dihydrohexosylceramides. To determine if 4-HPR inhibits dihydroceramide desaturase, we synthesized NBD-dihydroceramide and treated Hek293 cells with 4-HPR and analyzed the metabolites by HPLC. These analyses showed that NBD-dihydroceramide was taken up by the cells and converted to NBD-ceramides and more complex NBD-sphingolipids in control cells, however, within one hour of treatment with 10 ~{ and L~}M 4-HPR, the production of NBD-ceramide was blocked. In vitro assays of the desaturase using NBD-dihydroceramide also showed rapid and complete inhibition by 4-HPR. Interestingly, when Hek cells were treated with 4-HPR for one hour then the medium was changed, the recovery of dihydroceramide desaturase activity was very slow (i.e., t1/2 > 66 h); therefore, either 4-HPR is difficult to remove from cells or the inhibition is essentially irreversible. These findings establish that 4-HPR not only induces de novo sphingolipid biosynthesis but also inhibits dihydroceramide desaturase, resulting in production of abnormally high proportions of sphingolipids with dihydroceramide as the backbone. This raises the possibility that some of the effects of 4-HPR on cell behavior may be due to the presence of these abnormal species.

Amplification of the receptor tyrosine kinase ErbB-2 has been linked to the proliferation of breast cancer cells.1,2 Trastuzumab targets the extracellular domain of ErbB-2, leading to growth inhibition of approximately 15% of the breast cancers with genomic amplification of the ERBB2 gene.3 Clinical studies have demonstrated its efficacy in both early4 and metastatic breast cancers. 5,6 However, many tumors with ERBB2 amplification are not responsive to treatment.7 Moreover, the ones that initially respond, eventually progress and acquire drug resistance.8 An in vitro model for this acquired resistance was established by Chan & al.9 The breast cancer cell line, BT474, containing amplified ERBB2, was grown in the presence of trastuzumab for several months until subclones outgrew. Gene expression profiling was performed on these clones to determine differentially expressed genes between the parental and resistant cells. DARPP-32 (Dopamine and cAMP regulated phosphoprotein of 32kDa) was, by far, the most overexpressed transcript. DARPP-32 is coamplified with ERBB2 on the same amplicon of chromosome 17.10 This protein has been mostly described in neurobiology, but DARPP-32 overexpression was recently reported in gastrointestinal, esophageal, prostate and breast cancer.11 Therefore, we suggest that overexpression of DARPP-32 can cause acquired resistance of breast cancer cells to trastuzumab. The in vitro knockout of DARPP-32, using stable shRNA transfection, abolishes the resistance to trastuzumab in the clones, while overexpression of DARPP-32 in the parental cells results in de novo resistance. Overall, our results suggest that DARPP-32 may be a potential therapeutic target in breast cancer patients who develop acquired trastuzumab resistance.

Breast cancer is the most commonly diagnosed cancer in women. Despite recent advances in screening and early detection, breast cancer continues to result in a high incidence of morbidity and mortality. In its late stage the majority of patients exhibit signs of destructive skeletal metastasis. This complication is promoted by the production of growth factors by tumor cells which can induce tumor cell proliferation via their interaction with their respective receptors to initiate the vicious cycle of bone resorption. Inhibition of growth factors signaling through their receptors can therefore serve as a useful therapeutic approach to block bone metastasis. / The biological characteristics of cancer cells along with the targeting properties of immune system offer a novel approach in the treatment of breast cancer. Directed against HER-2/nue oncogene, the recombinant humanized monoclonal antibody, Trastuzumab (Herceptin), has shown significant clinical benefits for the treatment of HER-2 positive metastatic breast cancer. / In the present study, the effects of Herceptin and its molecular mechanism of action in abrogating the development and progression of osteolytic bone metastasis is investigated in an experimental mouse model of skeletal metastasis using human breast cancer cells BT-474 which are known to express high levels of HER-2. Treatment of BT-474 cells with Herceptin caused a dose dependent decrease in cell proliferation. In in vivo studies BT-474 cells were injected by into the left ventricle of female BALB/c nu/nu mice. Intraperitoneal infusion of Herceptin from the day of tumor cell inoculation or at the time of radiologically detectable skeletal metastasis either slowed the development or prevented the progression of skeletal metastasis as compared to control groups of animals receiving non-specific IgG. Bone histological analysis of long bones showed the ability of Herceptin to reduce the ratio of tumor volume to bone volume as well as mitotic index when Herceptin treatment was initiated from the day of tumor cell inoculation. Immunohistochemical analysis of long bones showed a significantly lower level of activated (phosphorylated) MAPK in bones of Herceptin treated animals. These studies demonstrate the ability of Herceptin to inhibit the development and abrogate the progression of skeletal metastasis associated with breast cancer by blocking the HER-2 mediated signaling pathways.

Among the myriad of naturally occurring bioactive compounds are the aminocyclopentitol-containing natural products that represent a family of sugar-derived microbial secondary metabolites, such as the antibiotics pactamycin, allosamidin, and trehazolin. Pactamycin, a structurally unique aminocyclitol antibiotic isolated from Streptomyces pactum, consists of a 5-membered ring aminocyclitol (cyclopentitol) unit, two aromatic rings (6-methylsalicylic acid (6-MSA) and 1-(3-Amino-phenyl)-ethanone or 3-aminoacetophenone) and a 1,1-dimethylurea. It has pronounced antibacterial, antitumor, antiviral, and antiplasmodial activities, but its development as a clinical drug was hampered by its broad cytotoxicity. Efforts to modulate its pharmacological and toxicity properties by structural modifications using synthetic organic chemistry have been difficult due to the complexity of its chemical structure. As part of our ongoing studies on the biosynthesis of aminocyclitol-derived bioactive natural products, we have identified the biosynthetic gene cluster of pactamycin in S. pactum ATCC 27456, which paves the way for a better understanding of pactamycin biosynthesis and generating novel pactamycin analogs through biosynthetic engineering. Through gene inactivations, feeding experiments, and in vitro enzymatic assay, we studied the biosynthesis of pactamycin, which include the modes of formation of the unique cyclopentitol unit, the 3-aminoacetophenone and the 6-methyl salicylic acid moieties. Armed with the tools needed to genetically engineer target strains of S. pactum, we were able to produce novel analogs of this untapped-class of natural products. TM-026 was generated from a ΔptmH (a radical SAM C-methyltransferase gene) mutant, whereas TM-025 was generated from a ΔptmH/ΔptmQ (a polyketide synthase gene) double knockout mutant. Both compounds show potent antimalarial activity, but lack significant antibacterial activity, and are about 10-30 times less toxic than pactamycin toward mammalian cells. The results suggest that distinct ribosomal binding selectivity or new mechanism(s) of action may be involved in their plasmodial growth inhibition, which may lead to the discovery of new antimalarial drugs and identification of new molecular targets within malarial parasites. TM-035 was also isolated from a ΔptmH mutant. However, we found that TM-035 showed no activity against bacteria, malarial parasites, and most tested mammalian cells, but it has potent growth inhibitory activity against two well-established human head and neck squamous cell carcinomas (SCC025 and SCC104) (IC₅₀ 725 nM) in an in vitro assay. More intriguingly, the compound is significantly less active against human primary epidermal keratinocytes (HPEK), demonstrating an interesting biological phenomenon and outstanding cell type selectivity, which may lead to the development of new anticancer chemotherapy. The production yield of pactamycin and its congeners under laboratory conditions is relatively low. This has hampered both mechanistic and preclinical studies of these promising compounds. To deepen our understanding of pactamycin biosynthesis and engineer mutant strains with improved production yields, we investigated pathway specific regulatory genes, ptmF and ptmE. Based on gene inactivation and RT-PCR studies, we found that the PtmF-PtmE system controls the transcription of the whole biosynthetic gene cluster. The results provide important insight into regulation of pactamycin biosynthesis and will contribute to future studies that aim at engineering high producing strains of S. pactum. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from Feb. 28, 2012 - Feb. 28, 2013