The ability of neoplastic cells to survive exposure to various chemotherapeutic drugs represents the main obstacle to successful cancer chemotherapy. These essays summarize the results of molecular biological, pharmacological, biochemical, cytogenetic, immunological, and pathological studies on mult

Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells

Dose-response studies were performed with the alkylating agents [nitrogen mustard, N,N'-bis(2-chloroethyl)-N-nitrosourea, melphalan, cisplatin (CDDP), 4-hydroperoxycyclophosphamide (4-HC), and trimethyleneiminethiophosphoramide] in both the MCF-7 human breast carcinoma cell line and the EMT6 and FSaIIC murine tumor lines Increasing selection pressure with the alkylating agents CDDP, melphalan, and 4-HC in vitro produced low levels (65- to 9-fold) of drug resistance, despite an intensive and prolonged treatment program The MCF-7 sublines made resistant to CDDP and 4-HC did not exhibit cross-resistance to other alkylating agents; however, the MCF-7 subline resistant to melphalan was partially cross-resistant to nitrogen mustard, 4-HC, and CDDP A log-linear relationship was maintained between surviving fraction of MCF-7 cells in culture and drug concentration with alkylating agents, whereas for nonalkylating agents the survival curves tended to plateau at high drug concentrations Log-linear tumor cell kill was also obtained over a wide dosage range with several alkylating agents in murine tumors treated in vivo Tumor cell survival assay by colony formation indicated the continuing importance of dose in the action of the drugs even at high levels of tumor cell kill With some agents, there was a difference between the slopes of the tumor cell killing curves in vivo as compared to in vitro Cyclophosphamide was far more potent in vitro (4-HC) than in vivo (cyclophosphamide) Trimethyleneiminethiophosphoramide and N,N'-bis(2-chloroethyl)-N-nitrosourea were both more potent in vivo than in vitro These differences may be explained by the various metabolic patterns of these drugs Dose of alkylating agents is clearly a crucial variable particularly where multilog tumor cell kill is the goal, and in this regard, the effect of drug dose on the tumoricidal action of the alkylating agents is substantially greater than for nonalkylating agents

/pdf/preclinical-studies-and-clinical-correlation-of-the-effect-27rw8vitxq.pdf

Preclinical studies and clinical correlation of the effect of alkylating dose.

P-glycoprotein (Pgp), the so-called multidrug transporter, is a plasma membrane glycoprotein often involved in the resistance of cancer cells towards multiple anticancer agents in the multidrug-resistant (MDR) phenotype. It has long been recognized that the lipid phase of the plasma membrane plays an important role with respect to multidrug resistance and Pgp because: the compounds involved in the MDR phenotype are hydrophobic and diffuse passively through the membrane; Pgp domains involved in drug binding are located within the putative transmembrane segments; Pgp activity is highly sensitive to its lipid environment; and Pgp may be involved in lipid trafficking and metabolism. Unraveling the different roles played by the membrane lipid phase in MDR is relevant, not only to the evaluation of the precise role of Pgp, but also to the understanding of the mechanism of action and function of Pgp. With this aim, I review the data from different fields (cancer research, medicinal chemistry, membrane biophysics, pharmaceutical research) concerning drug-membrane, as well as Pgp-membrane, interactions. It is emphasized that the lipid phase of the membrane cannot be overlooked while investigating the MDR phenotype. Taking into account these aspects should be useful in the search of ways to obviate MDR and could also be relevant to the study of other multidrug transporters.

Analysis of the tangled relationships between P-glycoprotein-mediated multidrug resistance and the lipid phase of the cell membrane.

Lipolytic Enzymes of the Gastrointestinal Tract- Kinetic Assay of Human Gastric Lipase on Short and Long Chain Triacylglycerol Emulsions- Inhibition of Pancreatic and Microbial Lipases by Proteins: Kinetic and Binding Studies- Tumor-Promoting Phorbol Diesters are Substrates for and Modulators of Diacylglycerol Lipase- Lipolytic Activities Operative at the Outer Surface of Rat Fat Cells- On the Mechanism of Endogenous Lipolysis in Rat Heart: A Role of Lysosomes ?- The Enzymes of Phosphatidylcholine Biosynthesis- Characterization of Choline and Ethanolamine Kinase Activities in Plasmodium-Infected Erythrocytes- Physiological Responses of Intestinal CTP: Phosphocholine Cytidylyltransferase and its Interaction with Lipophilic Drugs- Degradation of Membrane Phosphoglycerides by the Reversal of Phosphotransferase Reactions- Treatment of Rat Brain Microsomal Vesicles with Octyl-?-D-Glucopyranoside: A study on Ethanolamine Base-Exchange after Reaggregation- Effect of Modification of Membrane Phospholipid Composition on Phospholipid Methylation in Aggregating Cell Culture- Acetyltransferases and Transacylases Relative Rates in 1-Alkyl-or 1-Acyl-Phosphatidylcholine Synthesis by Rat Platelet Homogenates- Modulation of Deacylation-Reacylation Pathway of Phospholipid Metabolism and Turnover During Reperfusion of Ischemic Myocardium- Monoacylglycerol Acyltransferase: Stereospecificity and Evidence that the Hepatic and Intestinal Activities are Tissue-Specific Isoenzymes- The Interaction of Pancreatic Phospholipase A2 with Negatively Charged Substrates-Application: The Transformation of Soluble Phospholipase A2 into a Highly Penetrating "Membrane-Bound" Form- Mechanism of Interaction of Phospholipase A2 with Phospholipid Substrates and Activators- Comparison of the Activation of Soluble and Immobilized Phospholipase A2- Some Properties of Membrane-Bound Phospholipases A2- Endogenous Suppression of Neutral-Active and Calcium-Dependent Phospholipase A2 Activity in Human Polymorphonuclear Leukocytes- Properties of Pancreatic Phospholipases A1 and Intestinal Phospholipase A2 from Guinea Pig: Their Complementary Role in the Intestinal Absorption of Phospholipids- Partial Characterization of Cytosolic Phospholipase A1 of Rat Heart- Purification and Properties of Phospholipase A from the Outer Membrane of Overproducing Escherichia Coli K-12- Some Properties of Lysoplasmalogenase and Alkenylhydrolase from Rat Liver Microsomes- Purification of Platelet-Activating Factor Acetylhydrolase- Characterization of an Acetylhydrolase Isolated from Rat Alveolar Macrophages in Comparison with the Enzyme Present In Vivo in Lung Alveoli- Preliminary Study for High Performance Purification of a Hydrophobic Protein: A Biological Fluid Acetylhydrolase- Biosynthesis and Pharmacology of PAF-Acether (Platelet-Activating Factor)- The Peroxisomal Enzymes of Glycerolipid Metabolism- Deficiencies in Ether Glycerolipids and Their Biosynthesis in Inherited Peroxisomal Disorders- Impaired Maturation of Peroxisomal ?-Oxidation Enzymes in Fibroblasts from Patients with the Zellweger Syndrome and Infantile Refsum Disease- New Techniques in Glycosyltransferase Research- Biosynthesis of Gangliosides and Blood Group Glycolipids using Solubilized Glycosyltransferases- Solubilization of Lipid-Glycosyltransferases from Mitochondrial Outer Membranes- UDP-Glucose Sterol ?-Glucosyl Transferase, a Plant Sterol Conjugating Enzyme- Purification and Properties of Acid Sphingomyelinase from Human Urine- Heterogeneity of Human Sphingomyelinase: Relatedness of the Major Polypeptides- Immunological Studies on Acidic Sphingomyelinase- Immunological Studies on Lysosomal Sphingomyelinase: Immunization Procedures, Properties of Polyclonal and Monoclonal Antibodies Obtained and Effect of Triton X-100 on Binding of Enzyme Activity- ?-Glucocerebrosidase: Affinity Purification and Characterization of its Active Site with N-Alkyl Derivatives of L-Deoxynojirimycin- Human Acid ?-Glucosidase: Primary Structure of the Active Site- Specificity of Human Glucosylceramide ?-Glucosidase Towards Structurally Modified Glucosylceramides in a Liposomal Assay-System- Activator Proteins (Protein Cofactors) for the Catabolism of Glycosphingolipids- Glycosphingolipid Activator Proteins- Studies of SAP-1 and SAP-2 in Cultured Skin Fibroblasts- The Specificity of Cerebroside Sulfatase Activator- Two Heat-Stable Low-Molecular-Mass Proteins Stimulating the Enzymic Sphingomyelin Degradation Isolated from Human Gaucher and Normal Spleen- The Effects of Acidic Lipids and Heat-Stable Factor on the Physical-Chemical and Kinetic Properties of Glucocerebrosidase- A New Glucosylceramidase Activator in Human Placenta- Molecular Organization of Glycosphingolipids in Phosphatidylcholine Bilayers and Biological Membranes- Metabolic Incorporation of a New Fluorescent Anthracene Fatty Acid into the Membrane Lipids of Procaryotic and Eucaryotic Cells for Studying the Dynamic and Topology of Membranes- TMA-DPH as Specific Plasma Membrane Fluidity Probe for Intact Cells and Its Limitation- Phospholipid Bilayer Assembly: Facilitated Transmembrane Movement of Phosphatidylcholine- The Asymmetrical Distribution and Biosynthesis of Molecular Species of Phospholipids in Chick Brain Microsomes- Cholesterol Pools in Mycoplasma Membranes: Modifications in Phospholipid Composition Affect the Kinetics of Cholesterol Exchange with Lipid Vesicles- Structure and Function of the Nonspecific Lipid Transfer Protein (Sterol Carrier Protein 2)- A Model for Studying Membrane Fatty Acid Transport: Acyl-Coenzyme A Synthesis in Human Erythrocyte Ghosts- Transport of Fluorescent Fatty Acids into Cultured Cells: Analysis by the Fluorescence Activated Cell Sorter- Transport and Metabolism of Fatty Acids in Cultured Heart Muscle Cells from Neonatal Rats- Hormonal Regulation of Exogenous Fatty-Acid Incorporation into Lipids in Cultured Hamster Fibroblasts- Fatty Acid Composition and Metabolism of Tumor Cells Rendered Resistant to the Anticancer Drug Doxorubicin- HMGR (3-Hydroxy, 3-Methylglutaryl-CoA Reductase) Activity of Cultured Rat Brain Cells: Sensitivity to n-3 and n-6 Polyunsaturated Fatty Acids (PUFAs) from Cod-Liver and Sunflower Oils- Development and Maturation of Lipoprotein Lipase in Cultured Adipose Cells- Synthesis and Properties of Fluorescent Analogs of Cytidine Diphosphate-Diacylglycerol and Phosphatidylinositol- Biosynthesis and Intracellular Transport of Phospholipids in Yeast- Phospholipid Catabolism and Phospholipid Turnover in Cultured Cells- Aging of Rat Heart Myocytes and Fibroblasts: Relationship Between Lipid Composition, Membrane Organization and Biological Properties- Extracellular Fluid Viscosity: Its Role in the Regulation of Membrane Lipid Metabolism and Cellular Secretion- Control of Surface Sphingomyelinase Activity in Cultured Neuroblastoma Cells- Metabolism of Exogenous Gangliosides in Cultured Fibroblasts and Cerebellar Cells- Intracellular Aspects of Ganglioside Metabolism and Transport- Metabolic Aspects of Gangliosides involved in the Maturation of Cultured Nerve Cells- Regulation of Glycosphingolipid Anabolism in Fibroblasts by Ionophores, Plasma Membrane ATP-ASE Inhibiton, and Growth Factors- Nuclear Triiodothyronine Receptors and Mechanisms of Triiodothyronine and Insulin Action on the Synthesis of Cerebroside Sulfotransferase by Cultures of Cells Dissociated from Brains of Embryonic Mice- Utilization of Exogenous Ceramide for the Synthesis of Sphingolipids in Cultured SV40-Transformed Rat Schwann Cells- Genetic Regulation of Phospholipid Synthesis in Yeast- The Regulation of Arachidonic Acid Release in Parturition- Relationship Between Arachidonic Acid Biosynthesis and its Level in Rat Tissues- Regulatory Properties of Carnitine Palmitoyltransferase in the Mitochondrial Membrane of Liver- Structure and Regulation of the sn-Glycerol-3-Phosphate Acyltransferase of E Coli- Mammalian Phospholipase D and Related Activities- Metabolism of Inositol-Glycerophospholipids in Relation to Transmembrane Signalling and Calcium Mobilization- Protein Kinase C Regulation by Diacylglycerols: Structure-Function Relationships and Mechanism- Effect of Ischemia on Activities of Polyphosphoinositide Phosphodiesterase and Diacylglycerol Kinase in Gerbil Brain Synaptosomes- Receptor-Mediated Degradation of Choline Plasmalogens and Glycerophospho]ipid Methylation: A New Hypothesis- Transport and Processing of Lysosomal Enzymes- Genetic Heterogeneity of Gaucher Disease: Enzymatic and Immunologic Studies- Molecular Forms, Biosynthesis and Maturation of Glucocerebrosidase, a Membrane-Associated Lysosomal Enzyme Deficient in Gaucher Disease- Forms of Glucocerebrosidase Present in Tissues and Urine- The ?-Galactosidase-sialidase Complex: Studies on Normal and Mutant Human Fibroblasts- Intracellular Binding and Transport of Lysosomal Enzymes in Human and Bovine Tissues- Molecular Heterogeneity in O-Variant GM2 Gangliosidosis- Preliminary Characterization of a Ganglioside Sialidase in Normal and Mucolipidosis IV Fibroblasts- Sphingomyelinase and Niemann-Pick Disease- Fluorescent Derivatives of Sphingomyelin: Synthesis, Use as Substrates for Sphingomyelinase and for Diagnosis of Niemann-Pick Disease in Cultured Cells- Lipases, Cholesterylesterases and Carboxylesterases in Lymphoid Cell Lines: Substrate Specificity and Relation to Wolman's, Cholesteryl Ester Storage Diseases and Lipid Storage Myopathy- Use of Fluorescent Fatty Acids for Labelling Acid Lipase-Deficient Cells- Morphological and Biochemical Studies of Induced and Pathological Lipidosis in Cultured Muscle Cells- On the Composition of Autofluorescent Accumulation Products: Ceroid and Lipofuscin- Participants- Contributors

Enzymes of Lipid Metabolism II

Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles

We have studied the lipid composition and the acyl group composition, transport, and metabolism of doxorubicin-sensitive and -resistant rat glioblastoma cells in monolayer cultures (C6 clone). No difference in lipid composition was evidenced; the acyl group composition was, in contrast, highly modified in resistant cells, and these modifications appeared progressively during the acquisition of the resistance. Resistant cells were characterized by a decrease of n-9 eicosatrienoic acid and by a 2-3-fold increase of the proportions of the polyunsaturated fatty acids of the n-6 and n-3 families, especially arachidonic acid and n-3 docosahexaenoic acid. These differences were probably due to a 2-fold increase of the uptake of fatty acids by resistant cells as compared to sensitive cells, this increase allowing the suppression of an essential fatty acid deficiency. Only small changes in the transformations of 16 and 18-carbon atoms' fatty acids to higher analogues were evidenced. A small reduction of the desaturation of stearic acid to oleic acid and of linoleic acid to arachidonic acid was the main characteristic of resistant cells; these differences can be explained as a consequence of the suppression of the essential fatty acid deficiency.

https://cancerres.aacrjournals.org/content/canres/46/7/3258.full.pdf

Fatty Acid Composition Transport and Metabolism in Doxorubicin-sensitive and -resistant Rat Glioblastoma Cells

We have studied the plasma membrane fluidity of rat C6 glioblastoma cells and simian virus 40-transformed mouse liver cells in culture that had been rendered resistant to doxorubicin. This was done by the evaluation of fluorescence anisotropy of two probes; diphenylhexatriene was used on membrane microsomal fractions, and trimethylammonium-diphenylhexatriene was used on whole cell suspensions as a plasma membrane-specific probe since it does not enter the cells. A higher degree of membrane fluidity was exhibited with both techniques by doxorubicin-resistant glioblastoma cells as compared to the doxorubicin-sensitive strain, but in the transformed liver cells no such alteration was seen in the physical properties of their plasma membranes. A higher degree of acyl group unsaturation was noticed in the glioblastoma cells but not in the transformed liver cells upon acquisition of doxorubicin resistance. A similar simultaneous increase in acyl group unsaturation and membrane fluidity can be obtained easily by growing the sensitive cells with a medium supplemented with exogenous polyunsaturated fatty acids. This alteration does not modify the sensitivity of the cells to doxorubicin. We conclude from our work that the increase in membrane fluidity, which is frequently associated with drug resistance, is neither necessary nor sufficient for the expression of the resistance. The reason for a link between cell resistance to doxorubicin and plasma membrane fluidity remains to be found.

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Fluorescence Anisotropy of Cell Membranes of Doxorubicin-sensitive and -resistant Rodent Tumoral Cells

We have studied the incorporation and cytotoxicity of doxorubicin in cultured rat C6 glioma cells grown as monolayers. Net incorporation was linear up to high extracellular concentrations of drug (10 micrograms/ml). Cytotoxicity was evaluated both by tritiated thymidine incorporation inhibition and cloning efficiency inhibition. For similar total drug exposures, cytotoxicity was very different according to the exposure time and the exposure dose; incubation with a low dose for a long time was much less cytotoxic than that performed with a high dose for a short period of time. We have obtained several clones of doxorubicin-resistant cells. As compared to the wild strain, these cells were characterized by a larger size, a slower growth, a reduced cloning efficiency and a differential sensitivity of 100-1,000 to doxorubicin. Net incorporation of doxorubicin was 5-fold reduced in these cells, due to an increased efflux of the drug. These cells provide an interesting model of doxorubicin-resistant solid tumor in culture.

Cellular pharmacology of doxorubicin in sensitive and resistant rat glioblastoma cells in culture.

We have studied the metabolism of doxorubicin in rat and mouse liver, heart and hepatocytes. Doxorubicinol was present in all cases at very low levels and 7-deoxyaglycones were present only in extraphysiological conditions: no aglycones were found either in fresh livers or in hearts of animals treated with the drug, but they were produced in large amounts when the organs were left at room temperature after the death of the animal. Hepatocytes grown in primary cultures or hepatoma cells grown in continuous lines produced no 7-deoxyaglycones. Freshly isolated hepatocytes synthesized small amounts of 7-deoxyaglycones; however, when these hepatocytes were homogenized prior to incubation, high levels of 7-deoxyaglycones were produced. We conclude that 7-deoxyaglycone formation is possible only in injured tissue and is not, therefore, a normal pathway for doxorubicin.

Hepatic metabolism of doxorubicin in mice and rats

We have studied the growth inhibition, DNA synthesis inhibition and cell incorporation of the new anthracycline 4'-iodo-4'-deoxydoxorubicin (4'-iododoxorubicin) and of its 13-dihydroderivative in a model of doxorubicin-sensitive and -resistant rat C6 glioblastoma cells; results were compared to those obtained with doxorubicin and doxorubicinol in the same model. 4'-Iododoxorubicin was 7.5 times more potent than doxorubicin on the wild cell line and 45 times on the doxorubicin-resistant line, indicating that cross-resistance was only partial between the two drugs. Whereas doxorubicinol presented only a very faint cytotoxic activity, 4'-iododoxorubicinol retained the same activity as the parent drug against sensitive cells and a lower activity against resistant cells. DNA synthesis inhibition occurred for much higher doses than growth inhibition in the sensitive cells, but for similar doses in resistant cells. In both cell lines, 4'-iododoxorubicin and its metabolite were incorporated to a higher extent than doxorubicin and doxorubicinol respectively. Incorporation of metabolites was always lower than that of their parent compound. We have studied the metabolism of doxorubicin and 4'-iododoxorubicin by sensitive and resistant cells; only traces (less than 5%) of metabolites were identified in the cells as well as in the culture medium. A new cell line was selected for resistance in the presence of low amounts of 4'-iododoxorubicin. It presented a 6-fold resistance to 4'-iododoxorubicin and an 85-fold resistance to doxorubicin. Doxorubicin incorporation was markedly reduced in this cell line while 4'-iododoxorubicin was incorporated to the same extent as in the sensitive line. Measurements of drug efflux were performed in the three cell lines. No significant difference was exhibited between the efflux of doxorubicin and that of 4'-iododoxorubicin in each cell line; these effluxes were very rapid in the doxorubicin-selected resistant line, slow in the wild line and intermediate in the 4'-iododoxorubicin-selected line.

D Londos-Gagliardi

Papers

Fatty Acid Composition Transport and Metabolism in Doxorubicin-sensitive and -resistant Rat Glioblastoma Cells

Fluorescence Anisotropy of Cell Membranes of Doxorubicin-sensitive and -resistant Rodent Tumoral Cells

Cellular pharmacology of doxorubicin in sensitive and resistant rat glioblastoma cells in culture.

Hepatic metabolism of doxorubicin in mice and rats

Cellular pharmacology of 4'-iodo-4'-deoxydoxorubicin.