bafilomycin-a and Neoplasms

LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies.

Topics: Active Transport, Cell Nucleus; Antibodies; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Line, Tumor; Fatty Acids, Unsaturated; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Ivermectin; Macrolides; Membrane Proteins; Microtubule-Associated Proteins; Neoplasms; RNA Interference; RNA, Small Interfering

Many cell lines derived from tumors as well as transformed cell lines are far more sensitive to V-ATPase inhibitors than normal counterparts. The molecular mechanisms underlying these differences in sensitivity are not known. Using global gene expression data, we show that the most sensitive responses to HeLa cells to low doses of V-ATPase inhibitors involve genes responsive to decreasing intracellular iron or decreasing cholesterol and that sensitivity to iron uptake is an important determinant of V-ATPase sensitivity in several cancer cell lines. One of the most sensitive cell lines, melanoma derived SK-Mel-5, over-expresses the iron efflux transporter ferroportin and has decreased expression of proteins involved in iron uptake, suggesting that it actively suppresses cytoplasmic iron. SK-Mel-5 cells have increased production of reactive oxygen species and may be seeking to limit additional production of ROS by iron.

Topics: Antinematodal Agents; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cation Transport Proteins; Cell Line; Cell Line, Tumor; Cholesterol; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Iron; Macrolides; Neoplasms; Reactive Oxygen Species; Vacuolar Proton-Translocating ATPases

Successful cancer gene therapy depends on the development of non-toxic, efficient, tumor cell- specific systemic gene delivery systems. Our laboratory has developed a systemically administered, ligand-liposome complex that can effectively and preferentially deliver its therapeutic payload to both primary and metastatic tumors. To further improve the transfection efficiency of this targeting complex, a synthetic pH-sensitive histidylated oligolysine K[K(H)KKK]5-K(H)KKC (HoKC), designed to aid in endosomal escape and condensation of DNA, was included in the complex. The presence of HoKC increased the in vitro transfection efficiency over that of the original complex. Moreover, no increase in cytotoxicity was observed due to the presence of the HoKC peptide. In a DU145 human prostate cancer xenograft tumor model in athymic nude mice, inclusion of the HoKC peptide did not interfere with the tumor targeting specificity of the i.v. administered ligand/liposome/DNA complex. Most importantly, the level of transgene expression was significantly elevated in the tumors, but not in the normal tissue in those animals receiving the complex incorporating HoKC. The in vivo enhancement of transfection efficiency by this modified gene delivery vehicle could lead to a reduction in the number of administrations required for antitumor efficacy.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Combined Modality Therapy; DNA; Gene Expression; Genetic Therapy; Humans; Hydrogen-Ion Concentration; Immunoglobulin Fragments; Ligands; Liposomes; Macrolides; Male; Mice; Mice, Nude; Mitoxantrone; Neoplasms; Oligopeptides; Peptides; Prostatic Neoplasms; Receptors, Transferrin; Transfection; Xenograft Model Antitumor Assays