bafilomycin-a1 and Pancreatic-Neoplasms

Bafilomycin A1 is a specific inhibitor of vacuolar type proton pump (V-ATPase). This study was designed to examine the effect of bafilomycin A1 on the growth of Capan-1 human pancreatic cells which overexpress V-ATPase. Nude mice bearing a xenografted tumor of Capan-1 cell line were treated for 4 weeks with bafilomycin A1 (1.0 mg/kg/day). This treatment inhibited tumor growth, which was significantly reduced as compared with controls after 21 days (p < 0.05). However, there were no significant differences in body weights between groups. Microscopically, a large number of tumor cells in the treated group showed signs of apoptosis. These findings suggest that apoptosis induced by bafilomycin A1 was the event involved in suppression of tumor growth in vivo.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Enzyme Inhibitors; Humans; Macrolides; Mice; Mice, Nude; Neoplasm Transplantation; Pancreatic Neoplasms; Proton Pump Inhibitors; Tumor Cells, Cultured

Macroautophagy is a catabolic process through which redundant, aged, or damaged cellular structures are first enclosed within double-membrane vesicles (called autophagosomes), and thereafter degraded within lysosomes. Macroautophagy provides a primary route for the turnover of macromolecules, membranes and organelles, and as such plays a major role in cell homeostasis. As part of the stress response, autophagy is crucial to determine the cell fate in response to extracellular or intracellular injuries. Autophagy is involved in cancerogenesis and in cancer progression. Here we illustrate the essential methods for monitoring autophagy in pancreatic cancer cells.

Topics: Animals; Autophagosomes; Autophagy; Autophagy-Related Proteins; Carcinogenesis; Cell Culture Techniques; Cell Line, Tumor; Chloroquine; Disease Progression; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Humans; Immunoblotting; Lysosomes; Macrolides; Mice; Microscopy, Fluorescence; Pancreas; Pancreatic Neoplasms

Manzamine A, a member of the manzamine alkaloids, was originally isolated from marine sponges of the genus Haliclona. It was recently shown to have activity against pancreatic cancer cells, but the precise mechanism of action remained unclear. To further our understanding of the mechanism of action of manzamine A, chemogenomic profiling in the yeast S. cerevisiae was performed, suggesting that manzamine A is an uncoupler of vacuolar ATPases. Fluorescence microscopy confirmed this effect on yeast vacuoles, where manzamine A produced a phenotype very similar to that of the established v-ATPase inhibitor bafilomycin A1. In pancreatic cancer cells, 10 µM manzamine A affected vacuolar ATPase activity and significantly increased the level of autophagosome marker LC3-II and p62/SQSTM1 as observed by western blot analysis. Treatment with manzamine A in combination with bafilomycin A1 (inhibitor of autophagosome-lysosome fusion) did not change the levels of LC3-II when compared to cells treated with bafilomycin A1 alone, suggesting that manzamine A is a potential inhibitor of autophagy by preventing autophagosome turnover. As autophagy is essential for pancreatic tumor growth, blocking this pathway with manzamine A suggests a promising strategy for the treatment of pancreatic cancer.

Topics: Adaptor Proteins, Signal Transducing; Autophagy; Carbazoles; Cell Line, Tumor; Humans; Macrolides; Microtubule-Associated Proteins; Pancreatic Neoplasms; Proton Pumps; Protons; Saccharomyces cerevisiae; Sequestosome-1 Protein; Vacuolar Proton-Translocating ATPases; Vacuoles

Topics: Adenocarcinoma; Animals; Autophagy; Cell Line, Tumor; Cell Proliferation; Chloroquine; Humans; Immunoglobulin G; Macrolides; Melphalan; Mice; Oxidative Phosphorylation; Pancreatic Neoplasms; Reactive Oxygen Species; Xenograft Model Antitumor Assays

It has been known that the thermosensitivity of tumour cells can be increased by lowering intra-cellular pH (pHi) by inhibiting pHi control mechanisms. The pHi is partially controlled by transport of H+ from cytoplasm into endocytic and secretary systems in the cells mediated by vacuolar type H+ATPase and also by transport of H+ through plasma membrane.. This study investigated the effects the bafilomycine A1, an inhibitor of the vacuolar type H+ATPase and the EIPA, an inhibitor of the Na+/H+ exchanger in plasma membrane, on thermosensitivity of AsPC-1 cells, a human pancreatic cancer cell line. It also investigated the effects of combination of bafilomycine A1 and EIPA.. The treatment of cancer cells with bafilomycine A1 or EIPA individually slightly lowered pHi of the cells in vitro and increased the thermosensitivity of the cells.. The combination of these two drugs significantly lowered pHi and increased thermosensitivity of cancer cells in vitro and enhanced the heat-induced the growth delay of AsPC-1 tumours grown s.c in the legs of BALB/cA nude mice.

Topics: Amiloride; Animals; Antineoplastic Combined Chemotherapy Protocols; Body Temperature Regulation; Cell Line, Tumor; Cell Membrane; Combined Modality Therapy; Enzyme Inhibitors; Humans; Hydrogen-Ion Concentration; Hyperthermia, Induced; Macrolides; Male; Mice; Mice, Nude; Pancreatic Neoplasms; Proton-Translocating ATPases; Sodium-Hydrogen Exchangers; Transplantation, Heterologous

We have developed a potential tumor-targeting peptide vector (cRGD-hK) that is intended to be systemically and repeatedly administered to patients with advanced solid tumors. The peptide vector of 36 l-amino acid residues, CRGDCF(K[H-]KKK)6, comprises a tumor-homing RGD motif, a DNA-binding oligolysine, and histidyl residues to facilitate the delivery into the cytosol. Using cytomegalovirus-driven luciferase expression plasmids as a reporter, we tested the transfection efficiency of cRGD-hK in hepatoma and pancreatic cancer cell lines. Transfection with the cRGD-hK/plasmid complexes (molar ratio 4000:1) was inhibited by 50 nM bafilomycin A1, an inhibitor of the vacuolar ATPase endosomal proton pump, or 10 microM cycloRGDfV, an integrin alphavbeta3 antagonist, indicating that the three elements of cRGD-hK could function as expected, at least in vitro. In nude mice bearing tumors created by subcutaneous inoculation, luciferase activity in the tumor tissues 48 hours after the injection of the cRGD-hK/plasmid complexes through the tail vein (20 microg plasmids per mouse) was significantly higher than that in the lung, kidney, and spleen, but only slightly higher than that in the liver. Although the latter difference was small, we propose a potential nonviral gene therapy for advanced solid tumors through use of the tumor-targeting peptide vector.

Topics: Animals; Anti-Bacterial Agents; Enzyme Inhibitors; Genetic Therapy; Genetic Vectors; Histidine; Humans; Liver Neoplasms, Experimental; Luciferases; Macrolides; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Oligopeptides; Pancreatic Neoplasms; Plasmids; Polylysine; Proton-Translocating ATPases; Tissue Distribution; Tumor Cells, Cultured

Bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, can inhibit the growth of a variety of cultured cells in a dose-dependent manner, but its mechanism is unclear. The aim of this study was to examine whether bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis. The effect of bafilomycin A1 on tumour growth in vitro and in vivo was examined using an MTT assay and an in vivo tumour model. The presence or absence of apoptosis was determined by morphology and DNA analysis of tumour cells. The concentration of bafilomycin A1 for 50 per cent inhibition of cell viability during 72 h by the MTT assay was 5 nm. In DNA analysis, a ladder of fragmented DNA was detected in Capan-1 cells treated with bafilomycin A1 at concentrations greater than 10 nm for 24 h. Nude mice bearing a xenografted Capan-1 cell line tumour received 4 weeks of bafilomycin A1 (1.0 mg/kg per day). This treatment significantly inhibited tumour growth compared with controls after 21 days (P < 0.05). Histopathological examination of tumour cells in the treated group demonstrated signs of apoptosis with chromatin condensation and cell shrinkage. These observations suggest that bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis.

Topics: Adenocarcinoma; Adult; Animals; Anti-Bacterial Agents; Apoptosis; DNA Fragmentation; Dose-Response Relationship, Drug; Electrophoresis, Agar Gel; Enzyme Inhibitors; Humans; Macrolides; Male; Mice; Mice, Inbred BALB C; Pancreatic Neoplasms; Proton-Translocating ATPases; Tumor Cells, Cultured

To test whether in RINm5F rat insulinoma cells luminal acidity and the activity of a vacuolar-type proton pump are involved in calcium sequestration by intracellular calcium stores sensitive to inositol 1,4,5-trisphosphate (InsP3) we examined the effects of various proton-conducting ionophores and ammonium chloride, and of bafilomycin, a specific inhibitor of vacuolar proton pumps, on this parameter. Bafilomycin in concentrations up to 1 microM did not affect calcium sequestration by nonmitochondrial, InsP3-sensitive stores at all; 50 microM carbonylcyanide m-chlorophenylhydrazone, 50 microM monensin and 30 mM NH4Cl, which are diverse ways to dissipate transmembrane pH gradients, did not inhibit calcium sequestration. This argues against signficant involvement of internal acidity and vacuolar proton pumps in calcium sequestration by InsP3-sensitive stores in RINm5F cells. The proton-potassium-exchanging ionophore nigericin (20-100 microM), however, inhibited calcium sequestration by nonmitochondrial and InsP3-sensitive stores. This effect was dependent on the presence of potassium and could be reversed by inclusion of carbonylcyanide m-chlorophenylhydrazone or acetate in the incubation medium. Thus, the inhibitory effect of nigericin appears to be based on proton extrusion coupled to potassium influx across the membrane of calcium stores in RINm5F cells, creating an internal alkalinization of these stores. The effect of nigericin implies the continuous maintenance of an outside-to-inside potassium concentration gradient by nonmitochondrial calcium stores in RINm5F cells. This feature will be of potential interest in the identification of InsP3-sensitive calcium-storing organelles.

Topics: Ammonium Chloride; Animals; Anti-Bacterial Agents; Calcium; Cell Membrane; Cell Membrane Permeability; Endoplasmic Reticulum; Hydrogen-Ion Concentration; Inositol 1,4,5-Trisphosphate; Insulinoma; Ionophores; Islets of Langerhans; Liver; Macrolides; Nigericin; Pancreatic Neoplasms; Potassium; Proton Pump Inhibitors; Proton Pumps; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured; Vacuoles; Vanadates