Vijaya Damaraju - Academia.edu (original) (raw)

Papers by Vijaya Damaraju

Research paper thumbnail of Inhibition of nucleoside transporters by tyrosine kinase inhibitors and its effects on chemotherapy efficacy

Cancer Cell Microenvironment, Nov 25, 2014

Nucleoside transporters (NTs) are essential for transport of physiologic nucleosides and anticanc... more Nucleoside transporters (NTs) are essential for transport of physiologic nucleosides and anticancer nucleoside analogues. There are 7 NTs 5 of which play roles in cellular membrane transport namely human equilibrative nucleoside transporter 1 (hENT1), hENT2, human concentrative nucleoside transporter 1 (hCNT1), hCNT2, and hCNT3. Several studies have demonstrated roles for hENT1 in gemcitabine activity in pancreatic cancer where tumors that have low hENT1 levels have a poor response to gemcitabine compared to tumors with high hENT1 levels. Many clinical trial studies with tyrosine kinase inhibitors (TKIs) and a nucleoside analogue backbone have failed or been disappointing. Our group has discovered a possible explanation for the disappointing results of combination regimens consisting of a TKI and a nucleoside analogue. We have found that TKIs have an unappreciated pharmacologic effect in that TKIs are potent NT inhibitors. NT inhibitory properties may mean that it will be difficult to combine some TKIs with anticancer nucleoside drugs. Careful attention to scheduling TKIs and nucleosides may allow successful combinations of some TKIs and nucleoside anticancer drugs but for some other TKIs due to their long half-lives it may be impossible to successfully schedule them with nucleosides.

Research paper thumbnail of Autofluorescent fused-pyrimidine nucleosides: Synthesis and evaluation as permeants and inhibitors of human nucleoside transporters

Collection of Czechoslovak Chemical Communications, 2011

Research paper thumbnail of Chapter 6 Renal Conservation of Folates

Research paper thumbnail of L1210/B23.1 cells express equilibrative, inhibitor-sensitive nucleoside transport activity and lack two parental nucleoside transport activities

Journal of Biological Chemistry

Cultured mouse leukemia L1210 cells express the nucleoside-specific membrane transport processes ... more Cultured mouse leukemia L1210 cells express the nucleoside-specific membrane transport processes designated es, ei, and cif. The es and ei processes are equilibrative, but may be distinguished by the high sensitivity of the former to 6-[(4-nitrobenzyl)thio1-9-8-0-ribofuranosylpurine (NBMPR); the cif process is mediated by a Na+/nucleoside cotransporter of low sensitivity to NBMPR. Cells of an ei-deficient clonal line, L12 10/MC5-1, were mutagenized, and clones were selected in soft agar medium that contained (i) NBMPR (an inhibitor of es processes), (ii) erythro-9-(2-hydroxy-3-nony1)adenine (an inhibitor of adenosine deaminase), and (iii) arabinofuranosyladenine (a cytotoxic substrate for the three nucleoside transporters). The selection medium did not allow es activity and selected against cells that expressed the Na+-linked cif process. Cells of the L1210jB23.1 clonal isolate were deficient in cif transport activity, and inward fluxes of formycin B, a poorly metabolized analog of inosine, were virtually abolished by NBMPR in these cells. In the mutant cells, nonisotopic formycin B behaved as a countertransport substrate during influx of ['Hlformycin B, and inward fluxes of the latter were competitively inhibited by purine and pyrimidine nucleosides. The transport behavior of L121OjB23.1 cells indicates that (i) the mutationjselection procedure impaired or deleted the Na+-linked cif process and (ii) es nucleoside transport activity is expressed in the mutant cells.

Research paper thumbnail of 5′-S-(2-Aminoethyl)-N6-(4-nitrobenzyl)-5′-thioadenosine (SAENTA), a novel ligand with high affinity for polypeptides associated with nucleoside transporter

Biochemical Journal

Derivatives of N-(4-aminobenzyl)adenosine (substituted at the aminobenzyl group) and 5'-linked de... more Derivatives of N-(4-aminobenzyl)adenosine (substituted at the aminobenzyl group) and 5'-linked derivatives of N6-(4nitrobenzyl)adenosine (NBAdo) were evaluated as inhibitors of site-specific binding of [3H]nitrobenzylthioinosine (NBMPR) to pig erythrocyte membranes. Potent inhibitors were SAENTA [5'-S-(2-aminoethyl)-M-(4-nitrobenzyl)-5'thioadenosine] and acetyl-SAENTA (the 2-acetamidoethyl derivative of SAENTA). SAENTA was coupled to derivatized agarose-gel beads (Affi-Gel 10) to form an affinity matrix for chromatographic purification of NBMPR-binding polypeptides, which in pig erythrocytes are part of, or are associated with, the equilibrative nucleoside transporter. When pig erythrocyte membranes were solubilized with octyl glucoside (n-octyl ,-D-glucopyranoside) and applied to SAENTA-Affi-Gel 10 (SAENTA-AG10), polypeptides that migrated as a broad band on SDS/PAGE with an apparent molecular mass of 58-60 kDa were selectively retained by the affinity gel. These polypeptides were identified as components of the nucleoside transporter of pig erythrocytes by reactivity with a monoclonal antibody (mAb 1 IC4) that recognizes the NBMPR-binding protein of pig erythrocytes. Retention of the immunoreactive polypeptides by SAENTA-AGIO was blocked by NBAdo. The immunoreactive polypeptides were released from SAENTA-AG10 by elution under denaturing conditions with 1 % SDS or by elution with detergent solutions containing competitive ligands (NBAdo or NBMPR). A 72-fold enrichment of the immunoreactive polypeptides was achieved by a single passage of solubilized, protein-depleted membranes through a column of SAENTA-AG1O, followed by elution with detergent solutions containing NBAdo. These results demonstrate that polypeptide components of NBMPR-sensitive nucleoside-transport systems may be partly purified by affinity chromatography using gel media bearing SAENTA groups. grams during SDS/PAGE , have been identified as 55 kDa polypeptides by photoaffinity labelling with respectively [3H]NBMPR [10,13,14] and [3H]cytochalasin B . Separation of the nucleoside-and glucose-transporter polypeptides has been achieved by using antibodies to selectively remove the latter from human erythrocytic band-4.5 polypeptides, yielding preparations in which the protein content is more than 60% nucleoside transporter . An alternative approach to the preparation of nucleoside-transporter poly-Abbreviations used: NBMPR, nitrobenzylthioinosine {6-[(4-nitrobenzyl)thio]-9-(/8-D-ribofuranosyl)purine}; NBAdo, nitrobenzyladenosine [N6-(4nitrobenzyl)adenosine]; NBTGR, nitrobenzylthioguanosine {2-amino-6-[(4-nitrobenzyl)thio]-9-(f8-D-ribofuranosyl)purine}; SAENTA, 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine; acetyl-SAENTA, 5'-S-(2-acetamidoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine; SAENTA-AG10, SAENTA-Affi-Gel 10; mAb, monoclonal antibody; octyl glucoside, n-octyl ,-D-glucopyranoside; Tween 20, polyoxyethylene sorbitan monolaurate; IC50, concentration causing 50 % inhibition; PEI-cellulose, poly(ethylenimine)-cellulose; Tris 6.9 buffer, 50 mM-Tris/HCl buffer (pH 6.9, 22°C); Tris 7.4 buffer, 50 mM-Tris/HCl buffer (pH 7.4, 22°C). ¶ Present address:

Research paper thumbnail of Sodium-dependent nucleoside transport in mouse intestinal epithelial cells. Two transport systems with differing substrate specificities. J Biol Chem

Journal of Biological Chemistry

Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The upta... more Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The uptake of formycin By the C nucleoside analog of inosine, was concentrative and required extracellular sodium. The initial rate of sodium-dependent formycin B transport was saturable with a K,,, of 45 f 3 p~. The purine nucleosides adenosine, inosine, guanosine, and deoxyadenosine were all good inhibitors of sodium-dependent formycin B transport with 50% inhibition (ICSO) observed at concentrations less than 30 pM. Of the pyrimidine nucleosides examined, only uridine ( G O , 4 1 f 9 p~) was a good inhibitor. Thymidine and cytidine were poor inhibitors with ICao values greater than 300 p~. Direct measurements of ['Hlthymidine transport revealed, however, that the uptake of this nucleoside was also mediated by a sodium-dependent mechanism. Thymidine transport was inhibited by low concentrations of cytidine, uridine, adenosine, and deoxyadenosine (ICao values less than 25 pM), but not by formycin B, inosine, or guanosine (ICs0 values greater than 600 p~) .

Research paper thumbnail of A Uridine Glucuronosyltransferase 2B7 Polymorphism Predicts Epirubicin Clearance and Outcomes in Early-Stage Breast Cancer

Clinical breast cancer, Jan 25, 2015

Epirubicin is metabolized by uridine glucuronosyltransferase 2B7 (UGT2B7), an enzyme rich in sing... more Epirubicin is metabolized by uridine glucuronosyltransferase 2B7 (UGT2B7), an enzyme rich in single nucleotide polymorphisms (SNPs). We studied whether the -161 C > T germline SNP in UGT2B7 was related to epirubicin metabolism and whether differences exist in the toxicity and efficacy of epirubicin-based chemotherapy among patients who were TT homozygotes, CT heterozygotes, and CC homozygotes. A total of 132 women with non-metastatic breast cancer receiving FEC (5-fluorouracil 500 mg/m(2), epirubicin 100 mg/m(2), cyclophosphamide 500 mg/m(2)) were prospectively enrolled. Toxicity was assessed in cycle 1 using the National Cancer Institute Common Toxicity Criteria, version 2.0. The sequence at -161 was studied in 132 subjects; 37 were TT homozygotes, 63 were CT heterozygotes, 26 were CC homozygotes, and 6 could not be genotyped. The CC genotype patients had decreased epirubicin clearance (median, 103.3 L/hr) compared with the CT/TT genotype patients (median, 134.0 L/hr; P = .002)....

Research paper thumbnail of ChemInform Abstract: Autofluorescent Fused-Pyrimidine Nucleosides: Synthesis and Evaluation as Permeants and Inhibitors of Human Nucleoside Transporters

ChemInform, 2012

ABSTRACT Nucleosides with an aromatic five-membered ring heterocycle (N, O, or S) fused at C4-C5 ... more ABSTRACT Nucleosides with an aromatic five-membered ring heterocycle (N, O, or S) fused at C4-C5 of pyrimidin-2-one were prepared by ring closures with 5-(alkyn-1-yl)pyrimidin-2-one intermediates, heterocyclic atom replacements, and ring closure with a 5-aminocytidine derivative. Ultraviolet absorption and emission properties of the autofluorescent products enabled studies on permeation and inhibition of the trans-cellular trafficking effected by human equilibrative nucleoside transporters (hENTs). Some of the autofluorescent nucleosides were shown to be potent and selective inhibitors of human concentrative nucleoside transporters (hCNTs) in a companion study reported elsewhere.

Research paper thumbnail of Inhibition of sodium-independent and sodium-dependent nucleobase transport activities by tyrosine kinase inhibitors

Cancer chemotherapy and pharmacology, Jan 2, 2015

Effects of tyrosine kinase inhibitors (TKIs) on equilibrative nucleobase transport (ENBT) and sod... more Effects of tyrosine kinase inhibitors (TKIs) on equilibrative nucleobase transport (ENBT) and sodium-dependent nucleobase transport (SNBT) activities were investigated in normal human renal proximal tubule epithelial cells (hRPTECs) and in pig kidney cell line (LLC-PK1). Uptake assays were performed by assessing accumulation of radiolabeled nucleobases over time into hRPTECs or LLC-PK1 cell lines which express ENBT and SNBT activities, respectively. Dose-response curves for inhibition of 1 µM [(3)H]adenine or 1 µM [(3)H]hypoxanthine were examined in hRPTECs and in LLC-PK1 cells with varying TKI concentrations (0-100 µM) to calculate the IC50 values (mean ± S.E) for inhibition. Gefitinib inhibited ENBT activity with an IC50 value of 0.7 µM, thus indicating strong interactions of ENBT with gefitinib in hRPTECs. Erlotinib > sorafenib > imatinib > sunitinib inhibited ENBT with IC50 values of 15, 40, 60, 78 µM, respectively, whereas dasatinib, lapatinib, and vandetanib were not ...

Research paper thumbnail of Development of a new equation to estimate creatinine clearance in cancer patients

Cancer chemotherapy and pharmacology, Jan 19, 2015

Determining renal function is important for chemotherapy eligibility and dosing. Measured creatin... more Determining renal function is important for chemotherapy eligibility and dosing. Measured creatinine clearance (mCrCl) is the gold standard but is cumbersome. Equations estimating CrCl (eCrCl) based on serum creatinine (SCr) produce widely varying estimates. Considering that SCr is derived from skeletal muscle, this study prospectively developed a new eCrCl equation in cancer patients using CT-defined muscle surface area (MSA) and evaluated its utility in a separate, retrospective series. In a prospective, observational cohort study of cancer patients, mCrCl by 24-h urine collection was correlated with CT-determined MSA to create an equation for eCrCl [muscle surface area (cm(2)) × 42/SCr]. eCrCl by Wright, Cockcroft-Gault (CG), CKD-EPI, MDRD, and MSA was compared to mCrCl to determine fit. MSA-eCrCl was used to simulate carboplatin dosing in a retrospective series of advanced non-small cell lung cancer (NSCLC). Prospectively, 22 patients were accrued and evaluable (12 males; median...

Research paper thumbnail of Abstract 797: Interaction of multitargeted tyrosine kinase inhibitors with human nucleoside transporters

Research paper thumbnail of P8.04Comparison of Human Nucleoside Transporter Interactions with BCR-ABL Kinase Inhibitors

Annals of oncology : official journal of the European Society for Medical Oncology / ESMO, 2015

Human nucleoside transporters (hNTs) regulate cellular influx and disposition of anticancer nucle... more Human nucleoside transporters (hNTs) regulate cellular influx and disposition of anticancer nucleoside drugs. Cytarabine and fludarabine uptake in leukemic cells, which occurs primarily via human nucleoside transporter 1 (hENT1), was shown to be inhibited by imatinib and nilotinib [1, 2]. We studied interactions of the BCR-ABL kinase inhibitors bosutinib, dasatinib, imatinib, nilotinib and ponatinib with five human nucleoside transporters (hENT1-2 and hCNT1-3) that mediate cellular uptake of various anticancer nucleoside drugs. hNT interactions with bosutinib, dasatinib, imatinib, nilotinib and ponatinib were assessed by comparing their relative abilities to inhibit [3H]uridine uptake in yeast producing each of the recombinant hNTs individually. Inhibition of [3H]uridine, [3H]cytarabine, [3H]fludarabine and [3H]cladribine uptake and long-term accumulation was examined in the CEM cell line. Inhibition of [3H]uridine uptake was also studied in the K562 and KG1 cell lines. BCR-ABL TKIs...

Research paper thumbnail of Interactions of multitargeted kinase inhibitors and nucleoside drugs: achilles heel of combination therapy?

Molecular cancer therapeutics, 2015

Multitargeted tyrosine kinase inhibitors (TKI) axitinib, pazopanib, and sunitinib are used to tre... more Multitargeted tyrosine kinase inhibitors (TKI) axitinib, pazopanib, and sunitinib are used to treat many solid tumors. Combination trials of TKIs with gemcitabine, a nucleoside anticancer drug, in pancreas, renal, lung, ovarian, and other malignancies resulted in little benefit to patients. TKI interactions with human nucleoside transporters (hNT) were studied by assessing inhibition of [(3)H]uridine uptake in yeast producing recombinant hNTs individually and in cultured human cancer cell lines. Axitinib, pazopanib, and sunitinib inhibited hENT1 at low micromolar concentrations. In A549, AsPC-1, and Caki-1 cells, [(3)H]uridine, [(3)H]thymidine, [(3)H]gemcitabine, and [(3)H]fluorothymidine (FLT) accumulation was blocked by all three TKIs. Pazopanib > axitinib ≥ sunitinib inhibited hENT1 with IC50 values of 2, 7, and 29 μmol/L, respectively, leading to reduced intracellular gemcitabine and FLT accumulation. Pretreatment or cotreatment of Caki-1 cells with TKIs reduced cellular accu...

Research paper thumbnail of Up-regulation of P-glycoprotein confers acquired resistance to 6-mercaptopurine in human chronic myeloid leukemia cells

Oncology Letters, 2011

To investigate the mechanisms of cellular resistance to 6-mercaptopurine (6-MP) in chronic myeloi... more To investigate the mechanisms of cellular resistance to 6-mercaptopurine (6-MP) in chronic myeloid leukemia (CML), a 6-MP resistant cell line (K562-MP5) was established by stepwise selection of the CML cell line (K562). The results of the drug sensitivity analysis of the K562-MP5 cell line revealed the cells to be 339-fold more resistant to 6-MP compared with the parental K562 cells. K562-MP5 cells exhibited decreased accumulation and increased efflux of [ 14 C]6-MP and its metabolites. In addition, K562-MP5 cells showed increased [ 3 H]MTX transport. K562-MP5 cells over-expressed P-glycoprotein (P-gp) and up-regulated MDR1 mRNA levels. Taken together, these results suggest that the up-regulation of P-gp, which contributes to the decreased accumulation by increasing the efflux of 6-MP and its metabolites, underlies the mechanism of 6-MP resistance in K562 cells.

Research paper thumbnail of Chapter 6 Renal Conservation of Folates

Vitamins & Hormones, 2008

Folates play vital roles in one-carbon metabolism that produces the early substrates necessary fo... more Folates play vital roles in one-carbon metabolism that produces the early substrates necessary for nucleotide synthesis and salvage. Folates are essential vitamins in that humans cannot synthesize them and are totally dependent on the diet to obtain them. As water-soluble vitamins, they would be easily filtered by the kidney and lost to the tubular fluid but for a highly efficient renal conservation mechanism. This renal "folate trap" is made up of alpha-folate receptors and reduced folate carriers. The locations of these transporters are such that they direct folate transport from the apical/luminal sides of kidney cells to the basolateral/plasma sides. In addition, other transporters such as organic anion transporters and multidrug resistance proteins are also found in kidney cells and play a role in renal elimination of folate analogues such as antifolate cancer chemotherapy drugs. This chapter discusses how these transporter activities manifest themselves in folate and antifolate pharmacokinetics. It also discusses effects of alcohol on renal reabsorption of folates.

Research paper thumbnail of 173 Synergistic toxicity of tyrosine kinase inhibitors with Hsp90 inhibitor 17-AAG in lung cancer cell line

Ejc Supplements - EJC SUPPL, 2010

The ALK tyrosine kinase gene undergoes chromosomal rearrangements in the majority of Anaplastic L... more The ALK tyrosine kinase gene undergoes chromosomal rearrangements in the majority of Anaplastic Large Cell Lymphoma (ALCL) cases, and in a subset of Non Small Cell Lung Cancer (NSCLC), giving rise to various fusion proteins which bear a constitutively activated ALK kinase domain. Additionally, full length ALK is often found to be activated by gene amplification or by kinase domain point mutations in a significant fraction of neuroblastomas. In all three of these tumor types, there is strong evidence that activated ALK kinase is a driver oncogene, and that pharmacological intervention with small molecule inhibitors which target this kinase represents a promising therapeutic approach for affected patient populations. We have previously presented the identification of NMS-E628, an orally available small-molecule ALK kinase inhibitor. Here, we describe further preclinical characterization of this molecule. In vitro, NMS-E628 very selectively inhibited proliferation of ALK-dependent cell lines with IC50s in the sub 100 nM range. Interestingly, short-term exposure of the Karpas-299 ALK+ ALCL cell lines to NMS-E628 induced long lasting and profound induction of cell cycle block and inhibition of proliferation which was maintained for several days following withdrawal of the drug. Concomitant with this effect, sustained inhibition of NPM-ALK autophosphorylation and downstream signaling was observed, despite persistent expression of NPM-ALK fusion protein.

Research paper thumbnail of Nucleoside anticancer drugs: the role of nucleoside transporters in resistance to cancer chemotherapy†

Oncogene, 2003

The clinical efficacy of anticancer nucleoside drugs depends on a complex interplay of transporte... more The clinical efficacy of anticancer nucleoside drugs depends on a complex interplay of transporters mediating entry of nucleoside drugs into cells, efflux mechanisms that remove drugs from intracellular compartments and cellular metabolism to active metabolites. Nucleoside transporters (NTs) are important determinants for salvage of preformed nucleosides and mediated uptake of antimetabolite nucleoside drugs into target cells. The focus of this review is the two families of human nucleoside transporters (hENTs, hCNTs) and their role in transport of cytotoxic chemotherapeutic nucleoside drugs. Resistance to anticancer nucleoside drugs is a major clinical problem in which NTs have been implicated. Single nucleotide polymorphisms (SNPs) in drug transporters may contribute to interindividual variation in response to nucleoside drugs. In this review, we give an overview of the functional and molecular characteristics of human NTs and their potential role in resistance to nucleoside drugs and discuss the potential use of genetic polymorphism analyses for NTs to address drug resistance.

Research paper thumbnail of Role of Human Nucleoside Transporters in the Uptake and Cytotoxicity of Azacitidine and Decitabine

Nucleosides, Nucleotides and Nucleic Acids, 2012

The nucleoside analogs 5-azacytidine (azacitidine) and 5-aza-2&am... more The nucleoside analogs 5-azacytidine (azacitidine) and 5-aza-2'-deoxycytidine (decitabine) are active against acute myeloid leukemia and myelodysplastic syndromes. Cellular transport across membranes is crucial for uptake of these highly polar hydrophilic molecules. We assessed the ability of azacitidine, decitabine, and, for comparison, gemcitabine, to interact with human nucleoside transporters (hNTs) in Saccharomyces cerevisiae cells (hENT1/2, hCNT1/2/3) or Xenopus laevis oocytes (hENT3/4). All three drugs inhibited hCNT1/3 potently (K (i) values, 3-26 μM), hENT1/2 and hCNT2 weakly (K (i) values, 0.5-3.1 mM), and hENT3/4 poorly if at all. Rates of transport of [(3)H]gemcitabine, [(14)C]azacitidine, and [(3)H]decitabine observed in Xenopus oocytes expressing individual recombinant hNTs differed substantially. Cytotoxicity of azacitidine and decitabine was assessed in hNT-expressing or hNT-deficient cultured human cell lines in the absence or presence of transport inhibitors where available. The rank order of cytotoxic sensitivities (IC (50) values, μM) conferred by hNTs were hCNT1 (0.1) > hENT1 (0.3) ≫ hCNT2 (8.3), hENT2 (9.0) for azacitidine and hENT1 (0.3) > hCNT1 (0.8) ⋙ hENT2, hCNT2 (>100) for decitabine. Protection against cytotoxicity was…

Research paper thumbnail of Human Nucleoside Transporters: Biomarkers for Response to Nucleoside Drugs

Nucleosides, Nucleotides and Nucleic Acids, 2009

This review describes recent advances in developing human nucleoside transporters (hNTs) as bioma... more This review describes recent advances in developing human nucleoside transporters (hNTs) as biomarkers to predict response to nucleoside analog drugs with clinical activity. Understanding processes that contribute to drug response or lack thereof will provide strategies to potentiate efficacy or avoid toxicities of nucleoside analog drugs. hNT abundance, evaluated by immunohistochemical methods, has shown promise as a predictive marker to assess clinical drug response that could be used to identify patients who would most likely benefit from nucleoside analog drug treatment.

Research paper thumbnail of Characterization of Binding of Folates and Antifolates to Brush-Border Membrane Vesicles Isolated from Human Kidney

Molecular Pharmacology, 2004

Antifolates such as methotrexate, raltitrexed, and pemetrexed are among the most effective and wi... more Antifolates such as methotrexate, raltitrexed, and pemetrexed are among the most effective and widely used anticancer drugs. The antifolates are also among the most unpredictable of anticancer drugs with respect to pharmacokinetics and toxicity. In this study, we assessed the binding of folates and antifolates to the folate receptors (FRs) of human proximal tubules and the effects of pH on binding. Binding of [ 3 H]folic acid was pH-dependent, with maximal binding seen at pH 6. Equilibrium binding experiments with [ 3 H]folic acid showed that K d values were unaffected, and B max values increased as the pH was decreased from 8.0 to 6.0. Increasing the osmolarity at pH 6.0 had no effect on intravesicular content, confirming that increased site-specific binding caused the observed changes in B max values. Enzymatic cleavage of glycosyl-phosphatidyl-

Research paper thumbnail of Inhibition of nucleoside transporters by tyrosine kinase inhibitors and its effects on chemotherapy efficacy

Cancer Cell Microenvironment, Nov 25, 2014

Nucleoside transporters (NTs) are essential for transport of physiologic nucleosides and anticanc... more Nucleoside transporters (NTs) are essential for transport of physiologic nucleosides and anticancer nucleoside analogues. There are 7 NTs 5 of which play roles in cellular membrane transport namely human equilibrative nucleoside transporter 1 (hENT1), hENT2, human concentrative nucleoside transporter 1 (hCNT1), hCNT2, and hCNT3. Several studies have demonstrated roles for hENT1 in gemcitabine activity in pancreatic cancer where tumors that have low hENT1 levels have a poor response to gemcitabine compared to tumors with high hENT1 levels. Many clinical trial studies with tyrosine kinase inhibitors (TKIs) and a nucleoside analogue backbone have failed or been disappointing. Our group has discovered a possible explanation for the disappointing results of combination regimens consisting of a TKI and a nucleoside analogue. We have found that TKIs have an unappreciated pharmacologic effect in that TKIs are potent NT inhibitors. NT inhibitory properties may mean that it will be difficult to combine some TKIs with anticancer nucleoside drugs. Careful attention to scheduling TKIs and nucleosides may allow successful combinations of some TKIs and nucleoside anticancer drugs but for some other TKIs due to their long half-lives it may be impossible to successfully schedule them with nucleosides.

Research paper thumbnail of Autofluorescent fused-pyrimidine nucleosides: Synthesis and evaluation as permeants and inhibitors of human nucleoside transporters

Collection of Czechoslovak Chemical Communications, 2011

Research paper thumbnail of Chapter 6 Renal Conservation of Folates

Research paper thumbnail of L1210/B23.1 cells express equilibrative, inhibitor-sensitive nucleoside transport activity and lack two parental nucleoside transport activities

Journal of Biological Chemistry

Cultured mouse leukemia L1210 cells express the nucleoside-specific membrane transport processes ... more Cultured mouse leukemia L1210 cells express the nucleoside-specific membrane transport processes designated es, ei, and cif. The es and ei processes are equilibrative, but may be distinguished by the high sensitivity of the former to 6-[(4-nitrobenzyl)thio1-9-8-0-ribofuranosylpurine (NBMPR); the cif process is mediated by a Na+/nucleoside cotransporter of low sensitivity to NBMPR. Cells of an ei-deficient clonal line, L12 10/MC5-1, were mutagenized, and clones were selected in soft agar medium that contained (i) NBMPR (an inhibitor of es processes), (ii) erythro-9-(2-hydroxy-3-nony1)adenine (an inhibitor of adenosine deaminase), and (iii) arabinofuranosyladenine (a cytotoxic substrate for the three nucleoside transporters). The selection medium did not allow es activity and selected against cells that expressed the Na+-linked cif process. Cells of the L1210jB23.1 clonal isolate were deficient in cif transport activity, and inward fluxes of formycin B, a poorly metabolized analog of inosine, were virtually abolished by NBMPR in these cells. In the mutant cells, nonisotopic formycin B behaved as a countertransport substrate during influx of ['Hlformycin B, and inward fluxes of the latter were competitively inhibited by purine and pyrimidine nucleosides. The transport behavior of L121OjB23.1 cells indicates that (i) the mutationjselection procedure impaired or deleted the Na+-linked cif process and (ii) es nucleoside transport activity is expressed in the mutant cells.

Research paper thumbnail of 5′-S-(2-Aminoethyl)-N6-(4-nitrobenzyl)-5′-thioadenosine (SAENTA), a novel ligand with high affinity for polypeptides associated with nucleoside transporter

Biochemical Journal

Derivatives of N-(4-aminobenzyl)adenosine (substituted at the aminobenzyl group) and 5'-linked de... more Derivatives of N-(4-aminobenzyl)adenosine (substituted at the aminobenzyl group) and 5'-linked derivatives of N6-(4nitrobenzyl)adenosine (NBAdo) were evaluated as inhibitors of site-specific binding of [3H]nitrobenzylthioinosine (NBMPR) to pig erythrocyte membranes. Potent inhibitors were SAENTA [5'-S-(2-aminoethyl)-M-(4-nitrobenzyl)-5'thioadenosine] and acetyl-SAENTA (the 2-acetamidoethyl derivative of SAENTA). SAENTA was coupled to derivatized agarose-gel beads (Affi-Gel 10) to form an affinity matrix for chromatographic purification of NBMPR-binding polypeptides, which in pig erythrocytes are part of, or are associated with, the equilibrative nucleoside transporter. When pig erythrocyte membranes were solubilized with octyl glucoside (n-octyl ,-D-glucopyranoside) and applied to SAENTA-Affi-Gel 10 (SAENTA-AG10), polypeptides that migrated as a broad band on SDS/PAGE with an apparent molecular mass of 58-60 kDa were selectively retained by the affinity gel. These polypeptides were identified as components of the nucleoside transporter of pig erythrocytes by reactivity with a monoclonal antibody (mAb 1 IC4) that recognizes the NBMPR-binding protein of pig erythrocytes. Retention of the immunoreactive polypeptides by SAENTA-AGIO was blocked by NBAdo. The immunoreactive polypeptides were released from SAENTA-AG10 by elution under denaturing conditions with 1 % SDS or by elution with detergent solutions containing competitive ligands (NBAdo or NBMPR). A 72-fold enrichment of the immunoreactive polypeptides was achieved by a single passage of solubilized, protein-depleted membranes through a column of SAENTA-AG1O, followed by elution with detergent solutions containing NBAdo. These results demonstrate that polypeptide components of NBMPR-sensitive nucleoside-transport systems may be partly purified by affinity chromatography using gel media bearing SAENTA groups. grams during SDS/PAGE , have been identified as 55 kDa polypeptides by photoaffinity labelling with respectively [3H]NBMPR [10,13,14] and [3H]cytochalasin B . Separation of the nucleoside-and glucose-transporter polypeptides has been achieved by using antibodies to selectively remove the latter from human erythrocytic band-4.5 polypeptides, yielding preparations in which the protein content is more than 60% nucleoside transporter . An alternative approach to the preparation of nucleoside-transporter poly-Abbreviations used: NBMPR, nitrobenzylthioinosine {6-[(4-nitrobenzyl)thio]-9-(/8-D-ribofuranosyl)purine}; NBAdo, nitrobenzyladenosine [N6-(4nitrobenzyl)adenosine]; NBTGR, nitrobenzylthioguanosine {2-amino-6-[(4-nitrobenzyl)thio]-9-(f8-D-ribofuranosyl)purine}; SAENTA, 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine; acetyl-SAENTA, 5'-S-(2-acetamidoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine; SAENTA-AG10, SAENTA-Affi-Gel 10; mAb, monoclonal antibody; octyl glucoside, n-octyl ,-D-glucopyranoside; Tween 20, polyoxyethylene sorbitan monolaurate; IC50, concentration causing 50 % inhibition; PEI-cellulose, poly(ethylenimine)-cellulose; Tris 6.9 buffer, 50 mM-Tris/HCl buffer (pH 6.9, 22°C); Tris 7.4 buffer, 50 mM-Tris/HCl buffer (pH 7.4, 22°C). ¶ Present address:

Research paper thumbnail of Sodium-dependent nucleoside transport in mouse intestinal epithelial cells. Two transport systems with differing substrate specificities. J Biol Chem

Journal of Biological Chemistry

Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The upta... more Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The uptake of formycin By the C nucleoside analog of inosine, was concentrative and required extracellular sodium. The initial rate of sodium-dependent formycin B transport was saturable with a K,,, of 45 f 3 p~. The purine nucleosides adenosine, inosine, guanosine, and deoxyadenosine were all good inhibitors of sodium-dependent formycin B transport with 50% inhibition (ICSO) observed at concentrations less than 30 pM. Of the pyrimidine nucleosides examined, only uridine ( G O , 4 1 f 9 p~) was a good inhibitor. Thymidine and cytidine were poor inhibitors with ICao values greater than 300 p~. Direct measurements of ['Hlthymidine transport revealed, however, that the uptake of this nucleoside was also mediated by a sodium-dependent mechanism. Thymidine transport was inhibited by low concentrations of cytidine, uridine, adenosine, and deoxyadenosine (ICao values less than 25 pM), but not by formycin B, inosine, or guanosine (ICs0 values greater than 600 p~) .

Research paper thumbnail of A Uridine Glucuronosyltransferase 2B7 Polymorphism Predicts Epirubicin Clearance and Outcomes in Early-Stage Breast Cancer

Clinical breast cancer, Jan 25, 2015

Epirubicin is metabolized by uridine glucuronosyltransferase 2B7 (UGT2B7), an enzyme rich in sing... more Epirubicin is metabolized by uridine glucuronosyltransferase 2B7 (UGT2B7), an enzyme rich in single nucleotide polymorphisms (SNPs). We studied whether the -161 C > T germline SNP in UGT2B7 was related to epirubicin metabolism and whether differences exist in the toxicity and efficacy of epirubicin-based chemotherapy among patients who were TT homozygotes, CT heterozygotes, and CC homozygotes. A total of 132 women with non-metastatic breast cancer receiving FEC (5-fluorouracil 500 mg/m(2), epirubicin 100 mg/m(2), cyclophosphamide 500 mg/m(2)) were prospectively enrolled. Toxicity was assessed in cycle 1 using the National Cancer Institute Common Toxicity Criteria, version 2.0. The sequence at -161 was studied in 132 subjects; 37 were TT homozygotes, 63 were CT heterozygotes, 26 were CC homozygotes, and 6 could not be genotyped. The CC genotype patients had decreased epirubicin clearance (median, 103.3 L/hr) compared with the CT/TT genotype patients (median, 134.0 L/hr; P = .002)....

Research paper thumbnail of ChemInform Abstract: Autofluorescent Fused-Pyrimidine Nucleosides: Synthesis and Evaluation as Permeants and Inhibitors of Human Nucleoside Transporters

ChemInform, 2012

ABSTRACT Nucleosides with an aromatic five-membered ring heterocycle (N, O, or S) fused at C4-C5 ... more ABSTRACT Nucleosides with an aromatic five-membered ring heterocycle (N, O, or S) fused at C4-C5 of pyrimidin-2-one were prepared by ring closures with 5-(alkyn-1-yl)pyrimidin-2-one intermediates, heterocyclic atom replacements, and ring closure with a 5-aminocytidine derivative. Ultraviolet absorption and emission properties of the autofluorescent products enabled studies on permeation and inhibition of the trans-cellular trafficking effected by human equilibrative nucleoside transporters (hENTs). Some of the autofluorescent nucleosides were shown to be potent and selective inhibitors of human concentrative nucleoside transporters (hCNTs) in a companion study reported elsewhere.

Research paper thumbnail of Inhibition of sodium-independent and sodium-dependent nucleobase transport activities by tyrosine kinase inhibitors

Cancer chemotherapy and pharmacology, Jan 2, 2015

Effects of tyrosine kinase inhibitors (TKIs) on equilibrative nucleobase transport (ENBT) and sod... more Effects of tyrosine kinase inhibitors (TKIs) on equilibrative nucleobase transport (ENBT) and sodium-dependent nucleobase transport (SNBT) activities were investigated in normal human renal proximal tubule epithelial cells (hRPTECs) and in pig kidney cell line (LLC-PK1). Uptake assays were performed by assessing accumulation of radiolabeled nucleobases over time into hRPTECs or LLC-PK1 cell lines which express ENBT and SNBT activities, respectively. Dose-response curves for inhibition of 1 µM [(3)H]adenine or 1 µM [(3)H]hypoxanthine were examined in hRPTECs and in LLC-PK1 cells with varying TKI concentrations (0-100 µM) to calculate the IC50 values (mean ± S.E) for inhibition. Gefitinib inhibited ENBT activity with an IC50 value of 0.7 µM, thus indicating strong interactions of ENBT with gefitinib in hRPTECs. Erlotinib > sorafenib > imatinib > sunitinib inhibited ENBT with IC50 values of 15, 40, 60, 78 µM, respectively, whereas dasatinib, lapatinib, and vandetanib were not ...

Research paper thumbnail of Development of a new equation to estimate creatinine clearance in cancer patients

Cancer chemotherapy and pharmacology, Jan 19, 2015

Determining renal function is important for chemotherapy eligibility and dosing. Measured creatin... more Determining renal function is important for chemotherapy eligibility and dosing. Measured creatinine clearance (mCrCl) is the gold standard but is cumbersome. Equations estimating CrCl (eCrCl) based on serum creatinine (SCr) produce widely varying estimates. Considering that SCr is derived from skeletal muscle, this study prospectively developed a new eCrCl equation in cancer patients using CT-defined muscle surface area (MSA) and evaluated its utility in a separate, retrospective series. In a prospective, observational cohort study of cancer patients, mCrCl by 24-h urine collection was correlated with CT-determined MSA to create an equation for eCrCl [muscle surface area (cm(2)) × 42/SCr]. eCrCl by Wright, Cockcroft-Gault (CG), CKD-EPI, MDRD, and MSA was compared to mCrCl to determine fit. MSA-eCrCl was used to simulate carboplatin dosing in a retrospective series of advanced non-small cell lung cancer (NSCLC). Prospectively, 22 patients were accrued and evaluable (12 males; median...

Research paper thumbnail of Abstract 797: Interaction of multitargeted tyrosine kinase inhibitors with human nucleoside transporters

Research paper thumbnail of P8.04Comparison of Human Nucleoside Transporter Interactions with BCR-ABL Kinase Inhibitors

Annals of oncology : official journal of the European Society for Medical Oncology / ESMO, 2015

Human nucleoside transporters (hNTs) regulate cellular influx and disposition of anticancer nucle... more Human nucleoside transporters (hNTs) regulate cellular influx and disposition of anticancer nucleoside drugs. Cytarabine and fludarabine uptake in leukemic cells, which occurs primarily via human nucleoside transporter 1 (hENT1), was shown to be inhibited by imatinib and nilotinib [1, 2]. We studied interactions of the BCR-ABL kinase inhibitors bosutinib, dasatinib, imatinib, nilotinib and ponatinib with five human nucleoside transporters (hENT1-2 and hCNT1-3) that mediate cellular uptake of various anticancer nucleoside drugs. hNT interactions with bosutinib, dasatinib, imatinib, nilotinib and ponatinib were assessed by comparing their relative abilities to inhibit [3H]uridine uptake in yeast producing each of the recombinant hNTs individually. Inhibition of [3H]uridine, [3H]cytarabine, [3H]fludarabine and [3H]cladribine uptake and long-term accumulation was examined in the CEM cell line. Inhibition of [3H]uridine uptake was also studied in the K562 and KG1 cell lines. BCR-ABL TKIs...

Research paper thumbnail of Interactions of multitargeted kinase inhibitors and nucleoside drugs: achilles heel of combination therapy?

Molecular cancer therapeutics, 2015

Multitargeted tyrosine kinase inhibitors (TKI) axitinib, pazopanib, and sunitinib are used to tre... more Multitargeted tyrosine kinase inhibitors (TKI) axitinib, pazopanib, and sunitinib are used to treat many solid tumors. Combination trials of TKIs with gemcitabine, a nucleoside anticancer drug, in pancreas, renal, lung, ovarian, and other malignancies resulted in little benefit to patients. TKI interactions with human nucleoside transporters (hNT) were studied by assessing inhibition of [(3)H]uridine uptake in yeast producing recombinant hNTs individually and in cultured human cancer cell lines. Axitinib, pazopanib, and sunitinib inhibited hENT1 at low micromolar concentrations. In A549, AsPC-1, and Caki-1 cells, [(3)H]uridine, [(3)H]thymidine, [(3)H]gemcitabine, and [(3)H]fluorothymidine (FLT) accumulation was blocked by all three TKIs. Pazopanib > axitinib ≥ sunitinib inhibited hENT1 with IC50 values of 2, 7, and 29 μmol/L, respectively, leading to reduced intracellular gemcitabine and FLT accumulation. Pretreatment or cotreatment of Caki-1 cells with TKIs reduced cellular accu...

Research paper thumbnail of Up-regulation of P-glycoprotein confers acquired resistance to 6-mercaptopurine in human chronic myeloid leukemia cells

Oncology Letters, 2011

To investigate the mechanisms of cellular resistance to 6-mercaptopurine (6-MP) in chronic myeloi... more To investigate the mechanisms of cellular resistance to 6-mercaptopurine (6-MP) in chronic myeloid leukemia (CML), a 6-MP resistant cell line (K562-MP5) was established by stepwise selection of the CML cell line (K562). The results of the drug sensitivity analysis of the K562-MP5 cell line revealed the cells to be 339-fold more resistant to 6-MP compared with the parental K562 cells. K562-MP5 cells exhibited decreased accumulation and increased efflux of [ 14 C]6-MP and its metabolites. In addition, K562-MP5 cells showed increased [ 3 H]MTX transport. K562-MP5 cells over-expressed P-glycoprotein (P-gp) and up-regulated MDR1 mRNA levels. Taken together, these results suggest that the up-regulation of P-gp, which contributes to the decreased accumulation by increasing the efflux of 6-MP and its metabolites, underlies the mechanism of 6-MP resistance in K562 cells.

Research paper thumbnail of Chapter 6 Renal Conservation of Folates

Vitamins & Hormones, 2008

Folates play vital roles in one-carbon metabolism that produces the early substrates necessary fo... more Folates play vital roles in one-carbon metabolism that produces the early substrates necessary for nucleotide synthesis and salvage. Folates are essential vitamins in that humans cannot synthesize them and are totally dependent on the diet to obtain them. As water-soluble vitamins, they would be easily filtered by the kidney and lost to the tubular fluid but for a highly efficient renal conservation mechanism. This renal "folate trap" is made up of alpha-folate receptors and reduced folate carriers. The locations of these transporters are such that they direct folate transport from the apical/luminal sides of kidney cells to the basolateral/plasma sides. In addition, other transporters such as organic anion transporters and multidrug resistance proteins are also found in kidney cells and play a role in renal elimination of folate analogues such as antifolate cancer chemotherapy drugs. This chapter discusses how these transporter activities manifest themselves in folate and antifolate pharmacokinetics. It also discusses effects of alcohol on renal reabsorption of folates.

Research paper thumbnail of 173 Synergistic toxicity of tyrosine kinase inhibitors with Hsp90 inhibitor 17-AAG in lung cancer cell line

Ejc Supplements - EJC SUPPL, 2010

The ALK tyrosine kinase gene undergoes chromosomal rearrangements in the majority of Anaplastic L... more The ALK tyrosine kinase gene undergoes chromosomal rearrangements in the majority of Anaplastic Large Cell Lymphoma (ALCL) cases, and in a subset of Non Small Cell Lung Cancer (NSCLC), giving rise to various fusion proteins which bear a constitutively activated ALK kinase domain. Additionally, full length ALK is often found to be activated by gene amplification or by kinase domain point mutations in a significant fraction of neuroblastomas. In all three of these tumor types, there is strong evidence that activated ALK kinase is a driver oncogene, and that pharmacological intervention with small molecule inhibitors which target this kinase represents a promising therapeutic approach for affected patient populations. We have previously presented the identification of NMS-E628, an orally available small-molecule ALK kinase inhibitor. Here, we describe further preclinical characterization of this molecule. In vitro, NMS-E628 very selectively inhibited proliferation of ALK-dependent cell lines with IC50s in the sub 100 nM range. Interestingly, short-term exposure of the Karpas-299 ALK+ ALCL cell lines to NMS-E628 induced long lasting and profound induction of cell cycle block and inhibition of proliferation which was maintained for several days following withdrawal of the drug. Concomitant with this effect, sustained inhibition of NPM-ALK autophosphorylation and downstream signaling was observed, despite persistent expression of NPM-ALK fusion protein.

Research paper thumbnail of Nucleoside anticancer drugs: the role of nucleoside transporters in resistance to cancer chemotherapy†

Oncogene, 2003

The clinical efficacy of anticancer nucleoside drugs depends on a complex interplay of transporte... more The clinical efficacy of anticancer nucleoside drugs depends on a complex interplay of transporters mediating entry of nucleoside drugs into cells, efflux mechanisms that remove drugs from intracellular compartments and cellular metabolism to active metabolites. Nucleoside transporters (NTs) are important determinants for salvage of preformed nucleosides and mediated uptake of antimetabolite nucleoside drugs into target cells. The focus of this review is the two families of human nucleoside transporters (hENTs, hCNTs) and their role in transport of cytotoxic chemotherapeutic nucleoside drugs. Resistance to anticancer nucleoside drugs is a major clinical problem in which NTs have been implicated. Single nucleotide polymorphisms (SNPs) in drug transporters may contribute to interindividual variation in response to nucleoside drugs. In this review, we give an overview of the functional and molecular characteristics of human NTs and their potential role in resistance to nucleoside drugs and discuss the potential use of genetic polymorphism analyses for NTs to address drug resistance.

Research paper thumbnail of Role of Human Nucleoside Transporters in the Uptake and Cytotoxicity of Azacitidine and Decitabine

Nucleosides, Nucleotides and Nucleic Acids, 2012

The nucleoside analogs 5-azacytidine (azacitidine) and 5-aza-2&am... more The nucleoside analogs 5-azacytidine (azacitidine) and 5-aza-2'-deoxycytidine (decitabine) are active against acute myeloid leukemia and myelodysplastic syndromes. Cellular transport across membranes is crucial for uptake of these highly polar hydrophilic molecules. We assessed the ability of azacitidine, decitabine, and, for comparison, gemcitabine, to interact with human nucleoside transporters (hNTs) in Saccharomyces cerevisiae cells (hENT1/2, hCNT1/2/3) or Xenopus laevis oocytes (hENT3/4). All three drugs inhibited hCNT1/3 potently (K (i) values, 3-26 μM), hENT1/2 and hCNT2 weakly (K (i) values, 0.5-3.1 mM), and hENT3/4 poorly if at all. Rates of transport of [(3)H]gemcitabine, [(14)C]azacitidine, and [(3)H]decitabine observed in Xenopus oocytes expressing individual recombinant hNTs differed substantially. Cytotoxicity of azacitidine and decitabine was assessed in hNT-expressing or hNT-deficient cultured human cell lines in the absence or presence of transport inhibitors where available. The rank order of cytotoxic sensitivities (IC (50) values, μM) conferred by hNTs were hCNT1 (0.1) > hENT1 (0.3) ≫ hCNT2 (8.3), hENT2 (9.0) for azacitidine and hENT1 (0.3) > hCNT1 (0.8) ⋙ hENT2, hCNT2 (>100) for decitabine. Protection against cytotoxicity was…

Research paper thumbnail of Human Nucleoside Transporters: Biomarkers for Response to Nucleoside Drugs

Nucleosides, Nucleotides and Nucleic Acids, 2009

This review describes recent advances in developing human nucleoside transporters (hNTs) as bioma... more This review describes recent advances in developing human nucleoside transporters (hNTs) as biomarkers to predict response to nucleoside analog drugs with clinical activity. Understanding processes that contribute to drug response or lack thereof will provide strategies to potentiate efficacy or avoid toxicities of nucleoside analog drugs. hNT abundance, evaluated by immunohistochemical methods, has shown promise as a predictive marker to assess clinical drug response that could be used to identify patients who would most likely benefit from nucleoside analog drug treatment.

Research paper thumbnail of Characterization of Binding of Folates and Antifolates to Brush-Border Membrane Vesicles Isolated from Human Kidney

Molecular Pharmacology, 2004

Antifolates such as methotrexate, raltitrexed, and pemetrexed are among the most effective and wi... more Antifolates such as methotrexate, raltitrexed, and pemetrexed are among the most effective and widely used anticancer drugs. The antifolates are also among the most unpredictable of anticancer drugs with respect to pharmacokinetics and toxicity. In this study, we assessed the binding of folates and antifolates to the folate receptors (FRs) of human proximal tubules and the effects of pH on binding. Binding of [ 3 H]folic acid was pH-dependent, with maximal binding seen at pH 6. Equilibrium binding experiments with [ 3 H]folic acid showed that K d values were unaffected, and B max values increased as the pH was decreased from 8.0 to 6.0. Increasing the osmolarity at pH 6.0 had no effect on intravesicular content, confirming that increased site-specific binding caused the observed changes in B max values. Enzymatic cleavage of glycosyl-phosphatidyl-