thioinosine and Breast-Neoplasms

Topics: Animals; Breast Neoplasms; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Deficiency Diseases; Female; Fluorouracil; Guanine; Humans; Inosine Monophosphate; Lesch-Nyhan Syndrome; Leukemia; Nucleosides; Nucleotides; Sarcoma 180; Skin; Thioinosine

In this study we have examined the cytotoxic effects of different concentrations of adenosine (Ado) and deoxyadenosine (dAdo) on human breast cancer cell lines. Ado and dAdo alone had little effect on cell cytotoxicity. However, in the presence of adenosine deaminase (ADA) inhibitor, EHNA, adenosine and deoxyadenosine led to significant growth inhibition of cells of the lines tested. Ado/EHNA and dAdo/EHNA-induced cell death was significantly inhibited by NBTI, an inhibitor of nucleoside transport, and 5'-amino-5'-deoxyadenosine, an inhibitor of adenosine kinase, but the effects were not affected by 8-phenyltheophylline, a broad inhibitor of adenosine receptors. The Ado/EHNA combination brought about morphological changes consistent with apoptosis. Caspase-9 activation was observed in MCF-7 and MDA-MB468 human breast cancer cell lines on treatment with Ado/EHNA or dAdo/EHNA, but, as expected, caspase-3 activation was only observed in MDA-MB468 cells. The results of the study, thus, suggest that extracellular adenosine and deoxyadenosine induce apoptosis in both oestrogen receptor-positive (MCF-7) and also oestrogen receptor-negative (MDA-MB468) human breast cancer cells by its uptake into the cells and conversion to AMP (dAMP) followed by activation of nucleoside kinase, and finally by the activation of the mitochondrial/intrinsic apoptotic pathway.

Topics: Adenine; Adenosine; Apoptosis; Breast Neoplasms; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Cell Proliferation; Deoxyadenosines; Dose-Response Relationship, Drug; Humans; Receptors, Estrogen; Thioinosine; Time Factors

Gemcitabine and capecitabine are nucleoside analogues used in chemotherapy strategies for the treatment of breast cancer. We previously demonstrated that deficiency in hENT1, the most abundant and widely distributed plasma membrane nucleoside transporter in human cells, confers high-level resistance to gemcitabine toxicity in vitro, whereas the relationship between hENT1 activity and capecitabine toxicity is unknown. To determine the relationship between capecitabine cytotoxicity and hENT1 abundance, cultured MDA-MB-435s human mammary carcinoma cells were exposed to graded concentrations of the capecitabine metabolites, 5'-deoxy-5-fluorouridine or 5-fluorouracil, in the presence and absence of nitrobenzylmercaptopurine ribonucleoside (NBMPR), a tight-binding inhibitor of hENT1. The presence of NBMPR reduced the cytotoxic effects of 5'-deoxy-5-fluorouridine, indicating that hENT1 also enabled cellular uptake of this capecitabine metabolite by breast cancer cells. We report here the development of an immunohistochemical method to assess the hENT1 abundance of malignant cells in solid tumors. Frozen sections of 33 primary breast cancers were stained with monoclonal antibodies raised against a synthetic peptide derived from the large intracellular loop of hENT1, and staining intensity was scored on a 0-4+ scale. hENT1 staining intensity varied markedly among breast samples (4 with score 0, 5 with score 1+, 7 with score 2+, 14 with score 3+, 3 with score 4+), suggesting that at least 9 of the tumors were hENT1 deficient. We conclude that because hENT1 deficiency has previously been associated with nucleoside drug resistance, immunohistochemical staining of hENT1 warrants further study as a predictive tool for guiding the appropriate use of gemcitabine and capecitabine in the treatment of breast cancer.

Topics: Affinity Labels; Animals; Antibodies, Monoclonal; Antibody Formation; Antimetabolites, Antineoplastic; Breast Neoplasms; Capecitabine; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Deoxycytidine; Drug Resistance, Neoplasm; Equilibrative Nucleoside Transporter 1; Female; Floxuridine; Fluorouracil; Humans; Immunoenzyme Techniques; Membrane Transport Proteins; Mice; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasm Staging; Thioinosine

NB1011, a novel anticancer agent, targets tumor cells expressing high levels of thymidylate synthase (TS). NB1011 is converted intracellularly to bromovinyldeoxyuridine monophosphate (BVdUMP) which competes with the natural substrate, deoxyuridine monophosphate, for binding to TS. Unlike inhibitors, NB1011 becomes a reversible substrate for TS catalysis. Thus, TS retains activity and converts BVdUMP into cytotoxic product(s). In vitro cytotoxicity studies demonstrate NB1011's preferential activity against tumor cells expressing elevated TS protein levels. Additionally, NB1011 has antitumor activity in vivo. To identify drugs which interact synergistically with NB1011, we screened 13 combinations of chemotherapeutic agents with NB1011 in human tumor and normal cells. Dipyridamole and p-nitrobenzylthioinosine (NBMPR), potent inhibitors of equilibrative nucleoside transport, synergized with NB1011 selectively against 5-fluorouracil (5-FU)-resistant H630R10 colon carcinoma cells [combination index (CI)=0.75 and 0.35] and Tomudex-resistant MCF7TDX breast carcinoma cells (CI=0.51 and 0.57), both TS overexpressing cell lines. These agents produced no synergy with NB1011 in Det551 and CCD18co normal cells (CI > 1.1) lacking TS overexpression. Dipyridamole potentiated NB1011's cytotoxicity in medium lacking nucleosides and bases, suggesting a non-salvage-dependent mechanism. We demonstrate that nucleoside transport inhibitors, dipyridamole and NBMPR, show promise for clinically efficacious combination with NB1011.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Agents; Breast Neoplasms; Bromodeoxyuridine; Carrier Proteins; Cell Survival; Colonic Neoplasms; Dipyridamole; Drug Resistance, Neoplasm; Drug Synergism; Fluorouracil; Humans; Membrane Proteins; Nucleoside Transport Proteins; Quinazolines; Thioinosine; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured

MCF-7 cells display both nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative, but not the Na+-dependent, nucleoside transport. Transport of uridine by es is more sensitive to inhibition by purine nucleosides, whereas the ei component is more sensitive to nucleosides without an amino side group, such as inosine and thymidine. When exposed to 10 microM tamoxifen for 5 days, MCF-7 cells displayed a 44% decrease in the total number of NBMPR-binding sites [Bmax from 245000+/-18000 to 136000+/-25000 sites per cell (mean+/-S.E.M.; n=5; P<0.05)], and a 57% decrease in cell growth with no significant change in binding affinities [Kd from 0.37+/-0.05 to 0.45+/-0.08 nM (n=5; P>0.05)]. Kinetic studies of [3H]uridine transport showed a decrease in the Vmax of the es component from 21.7+/-0.3 (n=8) to 8.4 +/- 2.2 microM/s (n=4; P < 0.05), whereas the Vmax of the ei component [from 4.7 +/- 0.5 (n=8) to 5.8 +/- 1.6 microM/s (n=4; P > 0.05)] and Km values for both components [es from 460 +/- 80 to 630 +/- 280 microM (n>/=4; P > 0.05) and ei from 355 +/- 115 to 440 +/- 220 microM (n>/=4; P > 0.05)] did not change significantly. Oestradiol at 100 nM reversed almost completely the NBMPR-binding site decrease and growth retardation in tamoxifen-treated cells. Thus tamoxifen is shown to cause an oestrogen-reversible decrease of es nucleoside transporters in MCF-7 cells.

Topics: Binding Sites; Biological Transport; Breast Neoplasms; Carrier Proteins; Cell Division; Estradiol; Humans; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Protein Binding; Substrate Specificity; Tamoxifen; Thioinosine; Tumor Cells, Cultured; Uridine

Tamoxifen inhibits the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) to human MCF-7 breast cancer cells with an IC50 of 8 microM. Tamoxifen at 30 microM changed the apparent Kd for [3H]NBMPR binding from 0.63 +/- 0.12 to 4.75 +/- 0.58 nM, with little effect on the Bmax (311000 +/- 76000 and 263000 +/- 46000 sites per cell for untreated and tamoxifen-treated cells respectively). Corresponding to this decrease in binding of [3H]NBMPR in the presence of tamoxifen was an inhibition of NBMPR-sensitive equilibrative transport of 50 microM [3H]uridine (IC50 7-10 microM). In the presence of 15 microM tamoxifen, the apparent K(m) for [3H]uridine transport was increased from 390 +/- 30 to 1500 +/- 250 microM, with no change in Vmax (12.0 +/- 0.1 and 11.3 +/- 4.3 microM/s for untreated and tamoxifen-treated cells respectively). The inhibitory effect of tamoxifen on NBMPR-sensitive equilibrative uridine transport was specific, as similar results were also observed in HL-60 leukaemia and EL4 lymphoma cells. Furthermore a similar concentration of tamoxifen had no effect on the NBMPR-insensitive equilibrative transport of uridine in MCF-7, HL-60 and Morris 7777 hepatoma cells, and on the Na(+)-dependent transport of uridine in murine splenocytes. In this paper we demonstrate that tamoxifen by itself might have some antiproliferative effects through inhibition of DNA synthesis by blocking the nucleoside salvage pathway.

Topics: Animals; Breast Neoplasms; Carrier Proteins; Cells, Cultured; Humans; Kinetics; Membrane Proteins; Mice; Neoplasms; Nucleoside Transport Proteins; Nucleosides; Protein Binding; Sodium; Spleen; Tamoxifen; Thioinosine; Uridine