cytochrome-c-t and Carcinoma--Ductal--Breast

Cytochrome c (Cyt c) is a key protein that is needed to maintain life (respiration) and cell death (apoptosis). The dual-function of Cyt c comes from its capability to act as mitochondrial redox carrier that transfers electrons between the membrane-embedded complexes III and IV and to serve as a cytoplasmic apoptosis-triggering agent, activating the caspase cascade. However, the precise roles of Cyt c in mitochondria, cytoplasm and extracellular matrix under normal and pathological conditions are not completely understood. To date, no pathway of Cyt c release that results in caspase activation has been compellingly demonstrated in any invertebrate. The significance of mitochondrial dysfunctionality has not been studied in ductal carcinoma to the best of our knowledge. We used Raman spectroscopy and imaging to monitor changes in the redox state of the mitochondrial cytochromes in ex vivo surgically resected specimens of human breast tissues, and in vitro human breast cells of normal cells (MCF 10A), slightly malignant cells (MCF7) and highly aggressive cells (MDA-MB-231). We showed that Raman imaging provides insight into the biology of human breast ductal cancer. Here we show that proper concentration of monounsaturated fatty acids, saturated fatty acids, cardiolipin and Cyt c is critical in the correct breast ductal functioning and constitutes an important parameter to assess breast epithelial cells integrity and homeostasis. We look inside human breast ducts by Raman imaging answering fundamental questions about location and distribution of various biochemical components inside the lumen, epithelial cells of the duct and the extracellular matrix around the cancer duct during cancer development in situ. Our results show that human breast cancers demonstrate a redox imbalance compared to normal tissue. The reduced cytochrome c is upregulated in all stages of cancers development. The results of the paper shed light on a largely non-investigated issues regarding cytochromes and mitochondrial function in electron transfer chain. We found in histopathologically controlled breast cancer duct that Cyt c, cardiolipin, and palmitic acid are the main components inside the lumen of cancerous duct in situ. The presented results show direct evidence that Cyt c is released to the lumen from the epithelial cells in cancerous duct. In contrast the lumen in normal duct is empty and free of Cyt c. Our results demonstrate how Cyt c is likely to function in cancer developme

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Cytochromes c; Female; Humans; In Vitro Techniques; MCF-7 Cells; Mitochondria; Oxidation-Reduction; Spectrum Analysis, Raman

Growing interest in the beneficial effects of antioxidants has inspired the synthesis of new phenolic acid phenethyl ureas (PAPUs) with enhanced antioxidant potential. We have previously shown the capacity of one PAPU compound, (E)-1-(3,4-dihydroxyphenethyl)-3-styrylurea (PAPU1), to induce caspase-dependent apoptosis in melanoma cells. In the present study, we examined the anti-proliferative effects of PAPU compounds on MCF-7 human breast cancer cells and determined the molecular mechanisms involved. Treatment with PAPU compounds inhibited predominantly proliferation in these cells, where the PAPU1 was the most efficient form. Flow cytometric analysis showed that PAPU1 blocked cell cycle progression in the G(0)/G(1) phase, and reduced the proportion of cells in G(2)/M phase. This was related to the inhibition of cell cycle regulatory factors, including cyclin D/E and cyclin-dependent kinase (CDK) 2/4, through induction of p21(Cip1). PAPU1 also induced the mitochondrial-mediated and caspase-dependent apoptosis in MCF-7 cells. This was evidenced by cellular changes in the levels of Bcl-2 and Bax, loss of the mitochondrial membrane potential, release of cytochrome c into the cytosol, and caspase-9 activation. Collectively, our results suggest that G(1) cell cycle regulatory proteins and mitochondrial pathways are the crucial targets of PAPU1 in the chemoprevention of breast cancer cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma, Ductal, Breast; Caspases; Cell Proliferation; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cytochromes c; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Cells, Cultured; Urea

This work explores the use of 20-microm-i.d. polymeric polystyrene-divinylbenzene monolithic nanocapillary columns for the LC-ESI-MS analysis of tryptic digest peptide mixtures. In contrast to the packing of microparticles, capillary columns were prepared, without the need of high pressure, in fused-silica capillaries, by thermally induced in situ copolymerization of styrene and divinylbenzene. The polymerization conditions and mobile-phase composition were optimized for chromatographic performance leading to efficiencies over 100000 plates/m for peptide separations. High mass sensitivity (approximately 10 amol of peptides) in the MS and MS/MS modes using an ion trap MS was found, a factor of up to 20-fold improvement over 75-microm-i.d. nanocolumns. A wide linear dynamic range (approximately 4 orders of magnitude) was achieved, and good run-to-run and column-to-column reproducibility of isocratic and gradient elution separations were found. As samples, both model proteins and tissue extracts were employed. Gradient nano-LC-MS analysis of a proteolytic digest of a tissue extract, equivalent to a sample size of approximately 1000 cells injected, is presented.

Topics: Amino Acid Sequence; Animals; Carcinoma, Ductal, Breast; Caseins; Catalase; Cattle; Chickens; Chromatography, Liquid; Cytochromes c; Databases, Protein; Female; Horses; Humans; Lactoglobulins; Microscopy, Electron, Scanning; Molecular Sequence Data; Myoglobin; Neuropeptides; Ovalbumin; Peptide Fragments; Polystyrenes; Proteins; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Tissue Extracts; Trypsin; Trypsinogen