Biomedical subjects

Anna Maria Calcagno

Publications and source records attributed to Anna Maria Calcagno.

7 recordsLinked to original sources

PubMed2006

A "silent" polymorphism in the MDR1 gene changes substrate specificity.

Synonymous single-nucleotide polymorphisms (SNPs) do not produce altered coding sequences, and therefore they are not expected to change the function of the protein in which they occur. We report that a synonymous SNP in the Multidrug Resistance 1 (MDR1) gene, part of a haplotype previously linked to altered function of the MDR1 gene product P-glycoprotein (P-gp), nonetheless results in P-gp with altered drug and inhibitor interactions. Similar mRNA and protein levels, but altered conformations, were found for wild-type and polymorphic P-gp. We hypothesize that the presence of a rare codon, marked by the synonymous polymorphism, affects the timing of cotranslational folding and insertion of P-gp into the membrane, thereby altering the structure of substrate and inhibitor interaction sites.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Plasma membrane calcium ATPase (PMCA4): a housekeeper for RT-PCR relative quantification of polytopic membrane proteins.

BACKGROUND: Although relative quantification of real-time RT-PCR data can provide valuable information, one limitation remains the selection of an appropriate reference gene. No one gene has emerged as a universal reference gene and much debate surrounds some of the more commonly used reference genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At this time, no gene encoding for a plasma membrane protein serves as a reference gene, and relative quantification of plasma membrane proteins is performed with genes encoding soluble proteins, which differ greatly in quantity and in targeting and trafficking from plasma membrane proteins. In this work, our aim was to identify a housekeeping gene, ideally one that codes for a plasma membrane protein, whose expression remains the same regardless of drug treatment and across a wide range of tissues to be used for relative quantification of real-time RT-PCR data for ATP binding cassette (ABC) plasma membrane transporters. RESULTS: In studies evaluating the expression levels of two commonly used reference genes coding for soluble proteins and two genes coding for membrane proteins, one plasma membrane protein, plasma membrane calcium-ATPase 4 (PMCA4), was comparable to the two reference genes already in use. In addition, PMCA4 expression shows little variation across eight drug-treated cell lines and was found to be superior to GAPDH and HPRT1, commonly used reference genes. Finally, we show PMCA4 used as a reference gene for normalizing ABC transporter expression in a drug-resistant lung carcinoma cell line. CONCLUSION: We have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors.

ATP-Binding Cassette Transporters↗

PubMed2006

Role of the ABCG2 drug transporter in the resistance and oral bioavailability of a potent cyclin-dependent kinase/Aurora kinase inhibitor.

Cell cycle kinase inhibitors have advanced into clinical trials in oncology. One such molecule, JNJ-7706621, is a broad-spectrum inhibitor of the cyclin-dependent kinases and Aurora kinases that mediate G(2)-M arrest and inhibits tumor growth in xenograft models. To determine the putative mechanisms of resistance to JNJ-7706621 that might be encountered in the clinic, the human epithelial cervical carcinoma cell line (HeLa) was exposed to incrementally increasing concentrations of JNJ-7706621. The resulting resistant cell population, designated HeLa-6621, was 16-fold resistant to JNJ-7706621, cross-resistant to mitoxantrone (15-fold) and topotecan (6-fold), and exhibited reduced intracellular drug accumulation of JNJ-7706621. ABCG2 was highly overexpressed at both the mRNA ( approximately 163-fold) and protein levels. The functional role of ABCG2 in mediating resistance to JNJ-7706621 was consistent with the following findings: (a) an ABCG2 inhibitor, fumitremorgin C, restored the sensitivity of HeLa-6621 cells to JNJ-7706621 and to mitoxantrone; (b) human embryonic kidney-293 cells transfected with ABCG2 were resistant to both JNJ-7706621 and mitoxantrone; and (c) resistant cells that were removed from the drug for 12 weeks and reverted to susceptibility to JNJ-7706621 showed near-normal ABCG2 RNA levels. ABCG2 is likely to limit the bioavailability of JNJ-7706621 because oral administration of JNJ-7706621 to Bcrp (the murine homologue of ABCG2) knockout mice resulted in an increase in the plasma concentration of JNJ-7706621 compared with wild-type mice. These findings indicate that ABCG2 mediates the resistance to JNJ-7706621 and alters the absorption of the compound following administration.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2005

Modulatory effects of plant phenols on human multidrug-resistance proteins 1, 4 and 5 (ABCC1, 4 and 5).

Plant flavonoids are polyphenolic compounds, commonly found in vegetables, fruits and many food sources that form a significant portion of our diet. These compounds have been shown to interact with several ATP-binding cassette transporters that are linked with anticancer and antiviral drug resistance and, as such, may be beneficial in modulating drug resistance. This study investigates the interactions of six common polyphenols; quercetin, silymarin, resveratrol, naringenin, daidzein and hesperetin with the multidrug-resistance-associated proteins, MRP1, MRP4 and MRP5. At nontoxic concentrations, several of the polyphenols were able to modulate MRP1-, MRP4- and MRP5-mediated drug resistance, though to varying extents. The polyphenols also reversed resistance to NSC251820, a compound that appears to be a good substrate for MRP4, as predicted by data-mining studies. Furthermore, most of the polyphenols showed direct inhibition of MRP1-mediated [3H]dinitrophenyl S-glutathione and MRP4-mediated [3H]cGMP transport in inside-out vesicles prepared from human erythrocytes. Also, both quercetin and silymarin were found to inhibit MRP1-, MRP4- and MRP5-mediated transport from intact cells with high affinity. They also had significant effects on the ATPase activity of MRP1 and MRP4 without having any effect on [32P]8-azidoATP[alphaP] binding to these proteins. This suggests that these flavonoids most likely interact at the transporter's substrate-binding sites. Collectively, these results suggest that dietary flavonoids such as quercetin and silymarin can modulate transport activities of MRP1, -4 and -5. Such interactions could influence bioavailability of anticancer and antiviral drugs in vivo and thus, should be considered for increasing efficacy in drug therapies.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2004

Effects of an E-cadherin-derived peptide on the gene expression of Caco-2 cells.

PURPOSE: The goal of this study was to determine the effects of exposure to an HAV peptide (Ac-SHAVSS-NH2) on the protein and gene expression in Caco-2 cells, a model for the intestinal mucosa. METHODS: Caco-2 cells were incubated with either 100 or 500 microM of the hexapeptide then evaluated over a 48-h time period. RESULTS: Cell detachment from the monolayer was seen only after 48 h of exposure to the peptide, with the greatest effects occurring with a peptide concentration of 500 microM. Total protein expression of E-cadherin showed a decrease of nearly 20% at the 24-h time point for each concentration examined, whereas no significant changes were detected at the other time points studied. Short term exposure to a 500 microM solution of Ac-SHAVSS-NH2 caused few changes in gene expression as determined by Affymetrix GeneChip microarrays; however, longer exposure periods produced numerous changes in the treated cells. The variations in mRNA expression indicate that this HAV peptide has an effect in the E-cadherin signaling pathways. The greatest increases in mRNA expression were found in genes regulating excretion or degradation of the peptide. CONCLUSIONS: This work suggests that this HAV peptide produces effects that reach beyond modulation of adhesion.

Actins↗

PubMedDate not supplied

Modulation of cell adhesion molecules in various epithelial cell lines after treatment with PP2.

Regulation and expression of E-cadherin and other adhesion molecules were evaluated after exposure to a selective inhibitor of the Src family of tyrosine kinases and inducer of E-cadherin, PP2. E-cadherin is located within the intercellular junction, and it is involved in the management of paracellular permeability of various epithelial barriers in the body. Epithelial cell lines HCT-116, HT29, Caco-2, LS174T, and ARPE-19 were examined for morphological, functional, protein, and mRNA changes following 20 microM PP2 treatment. PP2 treatment caused cell clustering in Caco-2, HT29, and HCT-116 cells. E-cadherin also redistributed to the points of cell contact in Caco-2 cells. These changes suggest increased E-cadherin-dependent cell adhesion. Studies evaluating transepithelial electrical resistance, an established measurement of paracellular permeability, displayed increases in resistance for the Caco-2 cells following PP2 treatment, which correlates with our microscopy data. In addition, E-cadherin protein levels increased for all cells except HCT-116. ARPE-19 cells did not express E-cadherin at the protein or mRNA level. Expression of adhesion molecules varied for the cell lines, and only Claudin 3 mRNA expression was significantly increased in the three intestinal cell lines treated with PP2. Overall, our data suggest that E-cadherin is positively regulated by inhibition of Src tyrosine kinases at the functional and protein expression levels within these epithelial cell lines.

Adenocarcinoma↗

PubMedDate not supplied

Comparison of drug transporter levels in normal colon, colon cancer, and Caco-2 cells: impact on drug disposition and discovery.

A critical step in early phase drug development is the determination of oral bioavailability. In part, the ability to predict whether a drug will be effectively transported across the gastrointestinal mucosa can be estimated from the physicochemical properties of the compound. Although advancements through rational drug design have more correctly predicted bioavailability, considerable variability remains to be explained. Transporter expression throughout the gastrointestinal tract may explain much of this variation. ATP-binding cassette (ABC) transporters were the first family of transporters identified to modify bioavailability. More recently, the solute carrier family has also been shown to alter the pharmacokinetic profile of drugs. Currently, the Caco-2 human colon carcinoma cell line is often used by the pharmaceutical industry to evaluate intestinal absorption of drugs; however, in vivo/in vitro permeabilities with carrier mediated drugs do not correlate well, suggesting that Caco-2 transporter expression varies from that of the small intestine. With this is mind, we integrated U133A GeneChip expression data from the NCBIs Gene Expression Omnibus (GEO) collection and then compared the expression pattern of Caco-2 cells to normal colon to determine if the Caco-2 cell line is a reliable model for colonic delivery. Furthermore, transporter expression of Caco-2 cells was compared to that of human colon tumors to assess whether this cell line could be useful to predict drug absorption for colon cancer. Our analysis shows that the expression pattern for Caco-2 cells closely resembles the gene expression profile of transporters within the normal colon, suggesting that this cell line may serve as an in vitro model of colonic drug adsorption. However, the molecular "fingerprint" of Caco-2 was distinctly different from tumor samples, indicating that the Caco-2 model would unlikely predict accurate drug absorption for colon cancer sites.

ATP-Binding Cassette Transporters↗