monensin and Carcinoma--Squamous-Cell

Targeting the EGFR, with inhibitors such as erlotinib, represents a promising therapeutic option in advanced head and neck squamous cell carcinomas (HNSCC). However, they lack significant efficacy as single agents. Recently, we identified the ability of statins to induce synergistic cytotoxicity in HNSCC cells through targeting the activation and trafficking of the EGFR. However, in a phase I trial of rosuvastatin and erlotinib, statin-induced muscle pathology limited the usefulness of this approach. To overcome these toxicity limitations, we sought to uncover other potential combinations using a 1,200 compound screen of FDA-approved drugs. We identified monensin, a coccidial antibiotic, as synergistically enhancing the cytotoxicity of erlotinib in two cell line models of HNSCC, SCC9 and SCC25. Monensin treatment mimicked the inhibitory effects of statins on EGFR activation and downstream signaling. RNA-seq analysis of monensin-treated SCC25 cells demonstrated a wide array of cholesterol and lipid synthesis genes upregulated by this treatment similar to statin treatment. However, this pattern was not recapitulated in SCC9 cells as monensin specifically induced the expression of activation of transcription factor (ATF) 3, a key regulator of statin-induced apoptosis. This differential response was also demonstrated in monensin-treated ex vivo surgical tissues in which HMG-CoA reductase expression and ATF3 were either not induced, induced singly, or both induced together in a cohort of 10 patient samples, including four HNSCC. These results suggest the potential clinical utility of combining monensin with erlotinib in patients with HNSCC.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Drug Synergism; ErbB Receptors; Erlotinib Hydrochloride; Head and Neck Neoplasms; Humans; Monensin; Protein Kinase Inhibitors; Quinazolines; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

Ricin was encapsulated in various charged liposomes having 5 mol% PEG of different chain length on the surface. The cytotoxicity of ricin entrapped in these liposomal formulations was examined in human epidermoid carcinoma (KB) cells with a view to develop an optimum delivery system for ricin in vivo. It was observed that the cytotoxicity of ricin entrapped in various charged liposomes was significantly dependent on the surface charge as well as chain length of PEG. The maximum cytotoxicity of ricin was observed when it was delivered through negatively charged liposomes having 5 mol% PEG-2000 on the surface. Monensin enhances the cytotoxicity of ricin entrapped in various charged liposomes depending on the surface charge. Maximum potentiation of cytotoxicity of ricin was observed when it was delivered through negatively charged liposomes having 5 mol% PEG-2000 on the surface. Studies on the kinetics of inhibition of protein synthesis by ricin revealed that the lag period of inhibition of protein synthesis is significantly lengthened following its delivery through various charged liposomes. Monensin significantly reduced the lag period of action of ricin. It was also observed that the efficacies of monensin on the enhancement of cytotoxicity of ricin entrapped in various charged PEG-liposomes were highly related to their amount of cell association. The current study has demonstrated that by suitable adjustment of charge, density, and chain length of PEG on the surface of liposomes it would be possible to direct liposomal ricin to human tumor cells for their selective elimination in combination with monensin.

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cell Line, Tumor; Chemistry, Pharmaceutical; Drug Delivery Systems; Drug Interactions; Humans; KB Cells; Liposomes; Monensin; Polyethylene Glycols; Protein Synthesis Inhibitors; Ricin; Structure-Activity Relationship

Ricin was encapsulated into various sterically stabilized liposomes having different density of folate on the surface and the cytotoxicity of ricin in these liposomes was examined in KB cells. The effect of monensin in free and various sterically stabilized liposomal forms having different density of folate on the surface on the enhancement of cytotoxicity of ricin entrapped in these liposomes was also examined. It was observed that liposomal ricin having 0.5 mol% folate-PEG on the surface exhibits maximum cytotoxicity (IC(50)=1274 ng/ml) in KB cells as compared to non-targeted liposomes (IC(50)=3274 ng/ml). Monensin either in free form (266.2-fold) or liposomal form (291.5-fold) enhances the cytotoxicity of this targeted liposomal ricin significantly. This enhancement of the cytotoxicity of ricin entrapped in folate-targeted liposomes is further enhanced to 557.7-fold by monensin when it was delivered through folate-targeted (0.5 mol% folate-PEG) liposomes. The present study has clearly demonstrated that ricin entrapped in folate-tagged-sterically stabilized liposomes in combination with monensin intercalated in folate-tagged-sterically stabilized liposomes may have potential application for the treatment of cancer cells over-expressing folate receptors on the cell surface.

Topics: Animals; Carcinoma, Squamous Cell; Cells, Cultured; CHO Cells; Cricetinae; Cricetulus; Folic Acid; Folic Acid Transporters; Humans; Intercalating Agents; Liposomes; Monensin; Phosphatidylethanolamines; Polyethylene Glycols; Ricin

Parathyroid hormone-related protein (PTHrP) has been identified as a causative factor in the pathogenesis of humoral hypercalcemia of malignancy (HHM). The regulation and mechanisms of PTHrP secretion in most normal and malignant cells are unknown. PTHrP secretion, mRNA expression, and transcription were measured in neoplastic human squamous carcinoma cells (A253) and normal human foreskin keratinocytes (NHFK) by radioimmunoassay, RNase protection assay, and transient transfections of the 5'-flanking region of human PTHrP in a luciferase expression vector. Mechanisms of PTHrP secretion were investigated using chemicals (monensin, colchicine, cytochalasin B, guanosine 5'-[gamma-thio]triphosphate (GTPgammaS)) that interfere with or facilitate intracellular transport. Monensin inhibited PTHrP secretion in both NHFK and A253 cells. Ultrastructurally, monensin caused dilatation of rough endoplasmic reticulum and the formation of numerous cytoplasmic secretory vacuoles in both cell lines. Colchicine decreased PTHrP production in NHFK cells and stimulated PTHrP production and mRNA levels in A253 cells. Colchicine also stimulated transcription of the PTHrP-luciferase reporter gene. Cytochalasin B stimulated PTHrP secretion and mRNA expression in A253 cells, but had no effect in NHFK cells. GTPgammaS had no effect on PTHrP secretion in either cell line. It was concluded that PTHrP secretion is dependent on the constitutive movement of secretory vesicles to the cytoplasmic membrane and regulation of PTHrP secretion and mRNA expression are altered in squamous carcinoma cells compared to normal human keratinocytes in vitro.

Topics: Actin Cytoskeleton; Biological Transport; Carcinoma, Squamous Cell; Cells, Cultured; Colchicine; Cytochalasin B; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Keratinocytes; Microtubules; Monensin; Neoplasm Proteins; Parathyroid Hormone-Related Protein; Proteins; RNA, Messenger; Skin; Transcription, Genetic

This study characterized the cytosolic free Ca2+ concentration ([Ca2+]i) in NaCN-treated human A-431 cells. The resting [Ca2+]i was 85 +/- 8 nM (n = 141) in untreated cells at 37 degrees C, determined with the fura-2 fluorescence probe. When cells were treated with NaCN, [Ca2+]i increased in a time- and NaCN concentration-dependent manner. When cells were exposed to 10 mM NaCN for 10 min, [Ca2+]i increased 278 +/- 28% (n = 5) but returned to normal within 45 min after treatment. The [Ca2+]i increase depended on the presence of external Ca2+. La3+ and Cd2+, but not verapamil or nifedipine, inhibited the NaCN-induced [Ca2+]i increase. The NaCN-induced [Ca2+]i increase also depended on external Na+ (K1/2 = 85 mM). The intracellular Na+ concentration, measured with the fluorescence probe SBFI, increased 267 +/- 16% after NaCN treatment. The NaCN-induced [Ca2+]i increase was modulated by treatment with ouabain or veratridine and was completely blocked by tetrodotoxin, amiloride (K1/2 = 5.4 microM), and dichlorobenzamil (K1/2 = 0.28 microM). These results suggest NaCN activates the Na+/Ca2+ exchange system. TMB-8 and ryanodine both partially blocked the increase in [Ca2+]i in the presence of external Ca2+, indicating that Ca2+ release from intracellular pools also occurred after the initial Ca2+ influx. NaCN decreased inositol trisphosphates production. U-73122, bradykinin, or monensin did not prevent the NaCN-induced increase in [Ca2+]i. However, the magnitude of the [Ca2+]i increase caused by NaCN was abolished in ionomycin-treated the [Ca2+]i increase caused by NaCN was abolished in ionomycin-treated cells, indicating that intracellular Ca2+ release induced by NaCN is derived from an ionomycin-sensitive Ca2+ pool. The results suggest that NaCN initially increased Na+ influx, which activated the reverse mode of a Na+/Ca2+ exchanger, leading to an increase in Ca2+ influx. The Ca2+ influx induced a Ca2+ mobilization from only an ionomycin-sensitive intracellular Ca2+ pool containing ryanodine receptors.

Topics: Amiloride; Bradykinin; Calcium; Calcium Channels; Carcinoma, Squamous Cell; Carrier Proteins; Cytosol; Extracellular Space; Gallic Acid; Humans; Inositol Phosphates; Ion Channels; Ionomycin; Monensin; Muscle Proteins; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sodium; Sodium Cyanide; Sodium-Calcium Exchanger; Tumor Cells, Cultured

We have isolated brefeldin A (BFA)-resistant cell lines, KB/BF-1 and KB/BF-2, from the human epidermoid carcinoma KB cell line. The BFA-resistant phenotypes have been stably maintained for more than 3 months in the absence of BFA. KB/BF-1 and KB/BF-2 showed 10-30-fold higher resistance to cytotoxicity of BFA but were 2-3-fold more sensitive to monensin and nigericin, than KB cells. KB/BF-1 showed aberrant structures of the Golgi complex with poorly developed cisternae surrounded by many small vesicles. Immunocytochemical studies were done with antibodies against a Golgi-specific antigen (chronic rheumatoid arthritis antigen) and a coatomer subunit (beta-subunit for coat proteins of non-clathrin-coated vesicles). Golgi-specific markers were distributed into the small vesicles which were localized diffusedly in cytoplasm of KB/BF-1 cells. Such Golgi markers were observed in a strictly confined perinuclear region of the parental KB cells, whereas in the mutant cells the markers were distributed more diffusedly in dot-like structures at perinuclear regions. In addition, when exposed to BFA, the mutant and parental cells showed a different distribution of these markers. Synthesis and maturation of low density lipoprotein receptor showed apparently slower rates in processing of low density lipoprotein receptor in KB/BF-1 and KB/BF-2 cells than those observed in their parental KB cells. Protein secretion in KB/BF-1 and KB/BF-2 cells was about 30% less than that in KB cells. Much less inhibition by BFA on the secretion was observed in KB/BF-1 and KB/BF-2 cells. A BFA-resistant mutation in BFA-resistant KB cell lines appears to affect assembly of the Golgi apparatus as well as some Golgi-specific functions.

Topics: Brefeldin A; Carcinoma, Squamous Cell; Cyclopentanes; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Humans; Microscopy, Electron; Microscopy, Fluorescence; Monensin; Mutation; Precipitin Tests; Receptors, LDL; Tumor Cells, Cultured

The beta 2-adrenergic receptors of the human epidermoid carcinoma A431 cells reside on two polypeptide chains revealed by photoaffinity labelling with [125I]iodocyanopindolol-diazirine. These proteins correspond to two distinct populations of N-asparagine-linked glycoproteins: the 55-52 kDa molecules are associated with complex carbohydrate chain(s), the 65-63 kDa component with polymannosidic carbohydrate chain(s). Both types of receptors are present in preconfluent cells, but only the polymannosidic type is found in the postconfluent cells. Moreover, complex chains appear to be associated with the receptors with the highest affinity for (-)-isoproterenol and polymannosidic chains with the receptors with the lowest affinity for this agonist. the carbohydrate moiety of the beta-adrenergic receptor is involved in the expression and function of the beta 2-adrenergic receptors at the surface of the A431 cells, since tunicamycin and monensin, complete and partial inhibitors of glycosylation respectively, diminish the number of binding sites at the cell surface and increase the total number of sites in the cell. In these conditions a diminution of cyclic AMP accumulation is also observed.

Topics: Adrenergic beta-Antagonists; Binding Sites; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Chromatography, Affinity; Cyclic AMP; Glycosylation; Humans; Isoproterenol; Monensin; Oligosaccharides; Propanolamines; Receptors, Adrenergic, beta; Tunicamycin