bafilomycin-a and Breast-Neoplasms

The vacuolar (H(+))-ATPases (V-ATPases) are a family of ATP-driven proton pumps that couple ATP hydrolysis with translocation of protons across membranes. Previous studies have implicated V-ATPases in cancer cell invasion. It has been proposed that V-ATPases participate in invasion by localizing to the plasma membrane and causing acidification of the extracellular space. To test this hypothesis, we utilized two separate approaches to specifically inhibit plasma membrane V-ATPases. First, we stably transfected highly invasive MDA-MB231 cells with a V5-tagged construct of the membrane-embedded c subunit of the V-ATPase, allowing for extracellular expression of the V5 epitope. We evaluated the effect of addition of a monoclonal antibody directed against the V5 epitope on both V-ATPase-mediated proton translocation across the plasma membrane and invasion using an in vitro Matrigel assay. The addition of anti-V5 antibody resulted in acidification of the cytosol and a decrease in V-ATPase-dependent proton flux across the plasma membrane in transfected but not control (untransfected) cells. These results demonstrate that the anti-V5 antibody inhibits activity of plasma membrane V-ATPases in transfected cells. Addition of the anti-V5 antibody also inhibited in vitro invasion of transfected (but not untransfected) cells. Second, we utilized a biotin-conjugated form of the specific V-ATPase inhibitor bafilomycin. When bound to streptavidin, this compound cannot cross the plasma membrane. Addition of this compound to MDA-MB231 cells also inhibited in vitro invasion. These studies suggest that plasma membrane V-ATPases play an important role in invasion of breast cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Movement; Cytosol; Enzyme Inhibitors; Humans; Hydrogen-Ion Concentration; Ion Transport; Macrolides; Neoplasm Invasiveness; Protein Transport; Protons; Vacuolar Proton-Translocating ATPases

Dynamic interactions between intracellular networks regulate cellular homeostasis and responses to perturbations. Targeted therapy is aimed at perturbing oncogene addiction pathways in cancer, however, development of acquired resistance to these drugs is a significant clinical problem. A network-based computational analysis of global gene expression data from matched sensitive and acquired drug-resistant cells to lapatinib, an EGFR/ErbB2 inhibitor, revealed an increased expression of the glucose deprivation response network, including glucagon signaling, glucose uptake, gluconeogenesis and unfolded protein response in the resistant cells. Importantly, the glucose deprivation response markers correlated significantly with high clinical relapse rates in ErbB2-positive breast cancer patients. Further, forcing drug-sensitive cells into glucose deprivation rendered them more resistant to lapatinib. Using a chemical genomics bioinformatics mining of the CMAP database, we identified drugs that specifically target the glucose deprivation response networks to overcome the resistant phenotype and reduced survival of resistant cells. This study implicates the chronic activation of cellular compensatory networks in response to targeted therapy and suggests novel combinations targeting signaling and metabolic networks in tumors with acquired resistance.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Flow Cytometry; Gene Expression Profiling; Genomics; Glucose; Humans; Hypoglycemic Agents; Lapatinib; Macrolides; Metformin; Models, Biological; Molecular Targeted Therapy; Quinazolines; Receptor, ErbB-2; Signal Transduction

Indole-3-carbinol and its metabolic products are considered promising chemopreventive and anticancer agents. Previously we have shown that the indole-3-carbinol cyclic tetrameric derivative CTet induces autophagy and inhibits cell proliferation via inhibition of Akt activity and overexpression of p21/CDKN1A and GADD45A, in both estrogen receptor-positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cell lines. In the present study, we further characterize the autophagic response and investigate the mechanism through which CTet regulates these events.. Analysis of gene expression microarray data and subsequent confirmation by quantitative real-time PCR, showed that CTet is able to induce up-regulation of key signaling molecules involved in endoplasmic reticulum (ER) stress response (e.g. DDIT3/CHOP, CHAC1, ATF3, HSPA5/BiP/GRP78, CEBPB, ASNS) and autophagy (e.g. MAP1LC3B), in both MCF-7 and MDA-MB-231 cell lines. Moreover, the monitoring of Xbp-1 splicing confirmed the activation of IRE1/Xbp-1 ER stress response branch after CTet treatment. The role of autophagic processes (known to be induced by ER stress) was investigated further through ATG5 gene silencing and pharmacological inhibition of AVOs formation. CTet was shown to induce an autophagy-related cell death. Moreover, CTet-treated cells stained with Hoechst/PI revealed the presence of necrotic processes without evidence of apoptosis.. The ER stress response was identified as the main upstream molecular mechanism through which CTet acts in both hormone-responsive and triple-negative breast cancer cells. Because of its important role in cancer development, ER stress is a potential target in cancer therapy. The abiltiy of CTet to induce ER stress response and subsequently activate a death program in tumor cells confirms this molecule as a promising anticancer agent.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Indoles; Macrolides; Necrosis; Oligonucleotide Array Sequence Analysis; Proteasome Endopeptidase Complex; Reactive Oxygen Species; Regulatory Factor X Transcription Factors; Transcription Factors; X-Box Binding Protein 1

The basic drugs doxorubicin and mitoxantrone are known to be concentrated in acidic endosomes of cells. Here, we address the hypotheses that raising endosomal pH with the modifying agents chloroquine, omeprazole or bafilomycin A might decrease sequestration of anticancer drugs in endosomes, thereby increasing their cytotoxicity and availability for tissue penetration. Chloroquine, omeprazole and bafilomycin A showed concentration-dependent effects to raise endosomal pH, and to inhibit sequestration of doxorubicin in endosomes. Chloroquine and omeprazole but not bafilomycin A decreased the net uptake of doxorubicin into cells, but there was no significant effect on uptake of mitoxantrone. Omeprazole and bafilomycin A increased the cytotoxicity of the anticancer drugs for cultured cells, as measured in a clonogenic assay, whereas chloroquine had minimal effects on cytotoxicity despite reduced uptake of doxorubicin. Omeprazole but not chloroquine or bafilomycin A increased the penetration of anticancer drugs through multicellular layers of tumour tissue. We conclude that modifiers of endosomal pH might increase therapeutic effectiveness of basic drugs by increasing their toxicity and/or tissue penetration in solid tumours.

Topics: Animals; Antibiotics, Antineoplastic; Antimalarials; Antineoplastic Agents; Breast Neoplasms; Chloroquine; Doxorubicin; Drug Interactions; Endosomes; Enzyme Inhibitors; Hydrogen-Ion Concentration; Macrolides; Mammary Neoplasms, Animal; Mice; Mitoxantrone; Omeprazole; Sarcoma; Tumor Cells, Cultured