bafilomycin-a and Melanoma

Silvestrol is a cyclopenta[b]benzofuran that was isolated from the fruits and twigs of Aglaia foveolata, a plant indigenous to Borneo in Southeast Asia. The purpose of the current study was to determine if inhibition of protein synthesis caused by silvestrol triggers autophagy and apoptosis in cultured human cancer cells derived from solid tumors.. In vitro cell viability, flow cytometry, fluorescence microscopy, qPCR and immunoblot was used to study the mechanism of action of silvestrol in MDA-MB-435 melanoma cells.. By 24 h, a decrease in cyclin B and cyclin D expression was observed in silvestrol-treated cells relative to control. In addition, silvestrol blocked progression through the cell cycle at the G2-phase. In silvestrol-treated cells, DAPI staining of nuclear chromatin displayed nucleosomal fragments. Annexin V staining demonstrated an increase in apoptotic cells after silvestrol treatment. Silvestrol induced caspase-3 activation and apoptotic cell death in a time- and dose-dependent manner. Furthermore, both silvestrol and SAHA enhanced autophagosome formation in MDA-MB-435 cells. MDA-MB-435 cells responded to silvestrol treatment with accumulation of LC3-II and time-dependent p62 degradation. Bafilomycin A, an autophagy inhibitor, resulted in the accumulation of LC3 in cells treated with silvestrol. Silvestrol-mediated cell death was attenuated in ATG7-null mouse embryonic fibroblasts (MEFs) lacking a functional autophagy protein.. Silvestrol potently inhibits cell growth and induces cell death in human melanoma cells through induction of early autophagy and caspase-mediated apoptosis. Silvestrol represents a natural product scaffold that exhibits potent cytotoxic activity and could be used for the further study of autophagy and its relationship to apoptosis in cancer cells.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin B; Cyclin D; Gene Expression Regulation, Neoplastic; Humans; Macrolides; Melanoma; Mice; Triterpenes

We investigated whether alterations in the mechanisms involved in intracellular pH (pHin) and intracellular calcium ([Ca2+]in) homeostasis are associated with the metastatic potential of poorly (A375P) and highly (C8161) metastatic human melanoma cells. We monitored pHin and [Ca2+]in simultaneously, using the fluorescence of SNARF-1 and Fura-2, respectively. Our results indicated that steady-state pHin and [Ca2+]in between these cell types were not significantly different. Treatment of cells with NH4Cl resulted in larger pHin increases in highly than in poorly metastatic cells, suggesting that C8161 cells have a lower H+ buffering capacity than A375P. NH4Cl treatment also increased [Ca2+]in only in C8161 cells. To determine if the changes in [Ca2+]in triggered by NH4Cl treatment were due to alterations in either H+- or Ca2+-buffering capacity, cells were treated with the Ca2+-ionophore 4Br-A23187, to alter [Ca2+]in. The magnitude of the ionophore-induced [Ca2+]in increase was slightly greater in C8161 cells than in A375P. Moreover, A375P cells recover from the ionophore-induced [Ca2+]in load, whereas C8161 cells did not, suggesting that A375P may exhibit distinct [Ca2+]in regulatory mechanisms than C8161 cells, to recover from Ca2+ loads. Removal of extracellular Ca2+ ([Ca2+]ex) decreased [Ca2+]in in both cell types at the same extent. Ionophore treatment in the absence of [Ca2+]ex transiently increased [Ca2+]in in C8161, but not in A375P cells. Endoplasmic reticulum (ER) Ca2+-ATPase inhibitors such as cyclopiazonic acid (CPA) and thapsigargin (TG) increased steady-state [Ca2+]in only in C8161 cells. Together, these data suggest that the contribution of intracellular Ca2+ stores for [Ca2+]in homeostasis is greater in highly than in poorly metastatic cells. Bafilomycin treatment, to inhibit V-type H+-ATPases, corroborated our previous results that V-H+-ATPases are functionally expressed at the plasma membranes of highly metastatic, but not in poorly metastatic cells (Martínez-Zaguilán et al., 1993). Collectively, these data suggest that distinct pHin and [Ca2+]in regulatory mechanisms are present in poorly and highly metastatic human melanoma cells.

Topics: Ammonium Chloride; Anti-Bacterial Agents; Benzopyrans; Calcimycin; Calcium; Calcium-Transporting ATPases; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Homeostasis; Humans; Hydrogen-Ion Concentration; Indoles; Ionophores; Macrolides; Melanoma; Naphthols; Neoplasm Metastasis; Proton-Translocating ATPases; Rhodamines; Thapsigargin; Tumor Cells, Cultured

We have identified nerve growth factor receptor (NGFR) on the cell surface and a truncated nerve growth factor receptor (NGFRt) in the conditioned medium of NGFR-negative cells that have been transfected with either the gene or the cDNA for the full-length receptor. By using cell surface iodination or metabolic labeling of A875 human melanoma cells, coupled with immunoprecipitation, we have determined the half-life of the cell-associated receptor to be approximately 7 h. Concomitant with receptor degradation is the accumulation of NGFRt in the extracellular medium. Approximately one-fifth of the labeled receptor can be recovered as the truncated species. These data support the hypothesis that NGFRt is generated by proteolysis of previously intact receptor. Furthermore, although no specific protease inhibitor assayed could affect this processing, NGFR degradation and truncation were retarded by treatment with: 1) the weak base amines, ammonium chloride or methylamine; 2) the carboxylic ionophore, monensin; or 3) the vacuolar ATPase inhibitor, bafilomycin A1, all agents that dissipate endosomal/lysosomal proton gradients via alternate mechanisms. Incubation of cells at 4 degrees C precluded NGFR degradation and truncation. The presence of ligand did not alter the time course of receptor truncation.

Topics: Ammonium Chloride; Anti-Bacterial Agents; Blotting, Western; DNA; Electrophoresis, Polyacrylamide Gel; Endocytosis; Endopeptidases; Humans; Hydrolysis; Macrolides; Melanoma; Methylamines; Monensin; Precipitin Tests; Protease Inhibitors; Receptors, Cell Surface; Receptors, Nerve Growth Factor; Transfection; Tumor Cells, Cultured