Non-destructive and selective imaging of the functionally active, pro-invasive membrane type-1 matrix metalloproteinase (MT1-MMP) enzyme in cancer cells.

Proteolytic activity of cell surface-associated MT1-matrix metalloproteinase (MMP) (MMP-14) is directly related to cell migration, invasion, and metastasis. MT1-MMP is regulated as a proteinase by activation and conversion of the latent proenzyme into the active enzyme, and also via inhibition by tissue inhibitors of MMPs (TIMPs) and self-proteolysis. MT1-MMP is also regulated as a membrane protein through its internalization and recycling. Routine immunohistochemistry, flow cytometry, reverse transcription-PCR, and immunoblotting methodologies do not allow quantitative imaging and assessment of the cell-surface levels of the active, TIMP-free MT1-MMP enzyme. Here, we developed a fluorescent reporter prototype that targets the cellular active MT1-MMP enzyme alone. The reporter (MP-3653) represents a liposome tagged with a fluorochrome and functionalized with a PEG chain spacer linked to an inhibitory hydroxamate warhead. Our studies using the MP-3653 reporter and its inactive derivative demonstrated that MP-3653 can be efficiently used not only to visualize the trafficking of MT1-MMP through the cell compartment, but also to quantify the femtomolar range amounts of the cell surface-associated active MT1-MMP enzyme in multiple cancer cell types, including breast carcinoma, fibrosarcoma, and melanoma. Thus, the levels of the naturally expressed, fully functional, active cellular MT1-MMP enzyme are roughly equal to 1 × 10(5) molecules/cell, whereas these levels are in a 1 × 10(6) range in the cells with the enforced MT1-MMP expression. We suggest that the reporter we developed will contribute to the laboratory studies of MT1-MMP and then, ultimately, to the design of novel, more efficient prognostic approaches and personalized cancer therapies.

Roles of free radicals in type 1 phototherapeutic agents: aromatic amines, sulfenamides, and sulfenates.

Detailed analyses of the electron spin resonance (ESR) spectra, cell viability, and DNA degradation studies are presented for the photolyzed Type I phototherapeutic agents: aromatic amines, sulfenamides, and sulfenates. The ESR studies provided evidence that copious free radicals can be generated from these N-H, N-S, and S-O containing compounds upon photoirradiation with UV/visible light. The analyses of spectral data allowed us to identify the free radical species. The cell viability studies showed that these agents after exposure to light exert cytotoxicity to kill cancer cells (U937 leukemia cell lines HTC11, KB, and HT29 cell lines) in a dosage- and time-dependent manner. We examined a possible pathway of cell death via DNA degradation by a plasmid cleavage assay for several compounds. The effects of photosensitization with benzophenone in the presence of oxygen were examined. The studies indicate that planar tricyclic amines and sulfenamides tend to form π-electron delocalized aminyl radicals, whereas nonplanar ones tend to yield nitroxide radicals resulting from the recombination of aminyl radicals with oxygen. The ESR studies coupled with the results of cell viability measurements and DNA degradation reveal that planar N-centered radicals can provide higher potency in cell death and allow us to provide some insights on the reaction mechanisms. We also found the formation of azatropylium cations possessing high aromaticity derived from azepines can facilitate secondary electron transfer to form toxic O2(•-) radicals, which can further exert oxidative stress and cause cell death.

Type 1 Phototherapeutic Agents. 2. Cancer Cell Viability and ESR Studies of Tricyclic Diarylamines.

Type 1 phototherapeutic agents based on diarylamines were assessed for free radical generation and evaluated in vitro for cell death efficacy in the U937 leukemia cancer cell line. All of the compounds were found to produce copious free radicals upon photoexcitation with UV-A and/or UV-B light, as determined by electron spin resonance (ESR) spectroscopy. Among the diarylamines, the most potent compounds were acridan (4) and 9-phenylacridan (5), with IC50 values of 0.68 µM and 0.17 µM, respectively.

Type 1 phototherapeutic agents, part I: preparation and cancer cell viability studies of novel photolabile sulfenamides.

Novel type 1 phototherapeutic agents based on compounds containing S-N bonds (sulfenamides) were synthesized, assessed for free radical generation, and evaluated in vitro for cell death efficacy in four cancer cell lines (U937, HTC11, KB, and HT29). All of the compounds were found to produce copious free radicals upon photoexcitation with UV-A and/or UV-B light, as determined by electron spin resonance spectroscopy. Among the sulfenamides, the most potent compounds were derived from dibenzazepine 7b and dihydroacridine 8b as determined in all of the four cancer cell lines.