The chemotherapy of solid tumours is hampered by two major obstacles: (1) the lack of selectivity of the current drugs and (2) the reduced sensitivity to tumour drugs. Recent advances in cancer biology have led to the identification of novel molecular targets responsible for aggressive proliferation, drug resistance and invasiveness. Targeting one such factor, epidermal growth factor receptor (EGFR), has proven an effective strategy to block the progression of solid tumours. However, the potency of the current clinical drugs directed at EGFR is mitigated by their reversible cytostatic activity and reduced cytotoxicity. Here we describe the design and synthesis of a new class of agents termed "cascade release combi-molecule" (I-TZ), "programmed" to hammer the receptor by releasing multiple bioactive metabolites (I-TZ', I-TZ") in a step-wise fashion and a reactive DNA damaging species (TZ), with the purpose of promoting sustained inhibition of EGFR tyrosine kinase (TK) and drug-induced cytotoxicity. This thesis describes the synthesis of one such molecule, RB24 (I-TZ), and demonstrates that it was capable of being hydrolyzed to its various sub-components with an overall half-life of 42 min under physiological conditions. The I-TZ induced sustained inhibition of the EGFR TK and strong antiproliferative activity in human solid tumour cells. It also inflicted higher levels of DNA damage to cancer cells transfected with the EGFR or HER2 gene. This selectivity was based on a novel mechanism of targeting described as a bystander effect, whereby following distribution of the I-TZ in the perinuclear region, the alkylating species (TZ) was subsequently released towards the nucleus. The I-TZ was significantly more potent than its clinical counterpart, TemodaI(TM). Further investigation of the mechanism underlying its superior potency show that blockade of EGFR-mediated signaling led to down-regulation of: (1) MAPK mitogenic signaling, (2) the BER protein, XRCC1 and (3) anti-apoptotic signaling mediated by Bad. Further down-regulation of XRCCI, with a MEK1 inhibitor, led to the discovery of a significant synergistic antiproliferative effect and a novel formula to further enhance the potency of the I-TZ in refractory tumours. Furthermore, the in vivo activity of the cascade release principle was proven in a prostate cancer xenograft study using a more water-soluble analogue of RB24. Thus, we have developed a novel model for the therapy of growth factor receptor-expressing tumours from the design and synthesis of the probe drugs, to the elucidation of their mechanism of action and the demonstration of the in vivo efficacy of the proposed strategy.

Cer and So are involved in regulation of apoptosis and cell cycle arrest while on the other hand S1P promotes cell growth and inhibits apoptosis. The antagonistic effects of these metabolites are regulated by enzymes that interconvert Cer, So, and S1P. In this work two of these enzymes were studied: sphingosine phosphate lyase and acid ceramidase. First several methods to determine the activity of these enzymes were developed and optimized, resulting in the publication of sensitive fluorogenic and chromatographic methods for enzyme activity. Particularly the assay optimized for acid ceramidase activity was used in the finding and identification of new inhibitors in several compound libraries. As a result compounds RBM2-1B, RBM2-1D and RBM2-1E were identified as acid ceramidase and dihydroceramide desaturase inhibitors. Furthermore compounds RBM1-12, RBM1-13 and SABRAC were also described as acid ceramidase inhibitors. Since several publications described the upregulation of acid ceramidase in advanced prostate cancer, we decided to investigate the particular effect of the acid ceramidase inhibition in a cellular model of advanced prostate cancer. Using cells PC-3/Mc we inhibited acid ceramidase through two different approaches: first silencing the gen ASAH1 and comparing the effects with chemical inhibition of the enzyme using compounds RBM1-12, RBM1-13 and SABRAC. We evaluate the effect of acid ceramidase inhibition in cell growth, invasivity and 3D growth in vitro, finding a diminished growth and 3D growth in both cells those knockdown for ASAH1 and treated with inhibitors. Finally, the effect of ASAH1 silencing in in vivo tumor growth and lung colonization was also determined. To this end male NOD-SCID mice were used for xenotransplants with cells PC-3/Mc_ASAH1_KD or control and the tumor growth or lung colonization was followed by luminometry. We found that the silencing of ASAH1 in PC-3/Mc cells delayed the growth and also the lung colonization, highlighting the potential of acid ceramidase inhibition as adjuvant in the treatment of prostate cancer and also in the prevention of metastases formation.

No description available.

Exposure to ultraviolet radiation (UVR) and the familial melanoma susceptibility gene p16INK4A are among the major risk factors which have been identified to contribute to the development of melanoma. UVR can act as a mitogen to stimulate cell proliferation and as a genotoxin by causing DNA damage in skin. UVR is also known to activate the MAP kinase signaling pathways, including the c-JNK kinase, p38 MAP kinase and ERK signaling pathways. These MAP kinase pathways have been reported to regulate a number of downstream effectors involved in controlling transcription, translation, transcript stability, and protein activity and stability. These in turn determine the UVR induced cellular responses such as proliferation, differentiation or apoptosis. The gene locus p16INK4A encodes the protein p16 which functions as a negative regulator of cell cycle progression and as a tumor suppressor is found deleted or mutated in 10-30% of primary melanomas and > 80% of melanoma cell lines. Previous work has shown that a suberythemal dose of UVR induces the expression of p16 in both the A2058 melanoma cell line and human skin. The regulatory mechanism controlling the expression of p16 in response to UVR is currently unclear. This study aims to define the mechanism involved in UVR induced p16 expression. A cell line model was established using serum starved quiescent A2058 cells to mimic the quiescent cells at the basal layer of the epidermis. The physiological relevance of this in vitro model was then verified in vivo in the human skin model. Data from this study has shown that UVR induced ERK signaling regulates p16 expression at both the transcriptional and translational level. Transcriptional control of p16 expression was observed in irradiated quiescent A2058 cells, where an increase in p16 mRNA corresponded to the increase in p16 protein. This observation was validated in human skin. Two transcription factors known to regulate expression of p16INK4A, MiTF and JunB, are potential downstream targets of UVR induced ERK signaling. Data also suggested the possible involvement of other components contributing to the UVR induced p16 expression observed in through ERK signaling, as MiTF is only expressed by the melanocytes, and the staining pattern of JunB did not correlate with the patchy p16 protein staining observed in human skin. Evidence indicating translational control of p16 expression was noted in the irradiated non serum starved A2058 cells, where an increase in p16 protein was detected despite the lack of elevated p16 mRNA. Additional evidence for translational contol was observed in irradiated human skin, where the uniform p16 mRNA expression in the skin did not correspond to the patchy p16 protein staining. Data from this study suggests a biological connection between melanoma risk factors, UVR and p16 with other melanoma genes, BRAF, MC1R and MiTF. BRAF plays a vital role in the ERK signaling pathway. ERK can regulate MiTF activity and stability, and BMSH (a ligand for MC1R receptor) has been shown to potentiate the increase of p16 expression irradiated skin, indicating p16 expression could be regulated via MC1R signaling. Taken together, these results demonstrate that the suberythemal UVR induced p16 expression is regulated by the ERK signaling pathway at both the transcriptional and translational levels. Further work is required to provide a better understanding of the mechanism by which UVR activated ERK regulates p16 expression.

Thesis (Ed. D.)--University of Toronto, 2005.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005. / Also available in print.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 7 uppsatser.

Thesis (Ph.D.)--University of California, San Francisco, 2007. / Source: Dissertation Abstracts International, Volume: 68-04, Section: B, page: 2270. Adviser: Matthias Hebrok.

Thesis (Ph.D.)--University of California, San Francisco, 2010. / Source: Dissertation Abstracts International, Volume: 71-02, Section: B, page: . Adviser: Xin Chen.

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.