thioinosine and Colonic-Neoplasms

The combination of 2'-deoxyadenosine and 2'-deoxycoformycin is toxic for the human colon carcinoma cell line LoVo. In this study we investigated the mode of action of the two compounds and have found that they promote apoptosis. The examination by fluorescence microscopy of the cells treated with the combination revealed the characteristic morphology associated with apoptosis, such as chromatin condensation and nuclear fragmentation. The occurrence of apoptosis was also confirmed by the release of cytochrome c and the proteolytic processing of procaspase-3 in cells subjected to the treatment. To exert its triggering action on the apoptotic process, 2'-deoxyadenosine enters the cells through an equilibrative nitrobenzyl-thioinosine-insensitive carrier, and must be phosphorylated by intracellular kinases. Indeed, in the present work we demonstrate by analysis of the intracellular metabolic derivatives of 2'-deoxyadenosine that, as suggested by our previous findings, in the incubation performed with 2'-deoxyadenosine and 2'-deoxycoformycin, an appreciable amount of dATP was formed. Conversely, when also an inhibitor of adenosine kinase was added to the incubation mixture, dATP was not formed, and the toxic and apoptotic effect of the combination was completely reverted.

Topics: Antineoplastic Agents; Apoptosis; Caspases; Cell Adhesion; Cell Count; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Deoxyadenosines; Enzyme Precursors; Flow Cytometry; Humans; Microscopy, Fluorescence; Pentostatin; 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

We have investigated the hypothesis that adenosine, a purine nucleoside produced within hypoxic regions of solid tumors, may interfere with the recognition of tumor cells by cytolytic effector cells of the immune system. We measured the adhesion of murine spleen-derived anti-CD3-activated killer (AK) lymphocytes to syngeneic MCA-38 colon adenocarcinoma cells in a model system. Adenosine, in the presence of the adenosine deaminase inhibitor coformycin to prevent the breakdown of adenosine, inhibited adhesion by up to 60%. The inhibitory effect of adenosine was exerted on the AK cells and not on the MCA-38 targets. The response to adenosine was generated at the cell surface, since the inhibition of adhesion was not abrogated by S-(4-nitrobenzyl)-6-thioinosine or dipyridamole, which block adenosine uptake. The inhibition of adhesion due to adenosine was not blocked by either the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or the A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine. This suggested that a non-A1, A2 receptor might be involved. The relative order of potencies of adenosine and common analogues was: 5'-N-ethylcarboxamidoadenosine = adenosine = (R)-phenylisopropyladenosine > N6-cyclopentyladenosine > 2-chloro-N6-cyclopentyladenosine = 2-p-(2- carboxyethyl)phenethylamino-5'-N-ethyl-carboxamidoadenosine. This agonist potency profile was again inconsistent with either the A1 or the A2 receptor subtype but indicated that the recently described A3 receptor subtype might be responsible for the inhibition of adhesion. Consistent with this suggestion, aminophenylethyladenosine, an adenosine analogue that binds with high affinity to A3 receptors, inhibited the adhesion of AK cells to MCA-38 tumor cells with high potency (50% inhibitory concentration approximately 1 nM). Adenosine, therefore, interferes with the AK cell recognition of colorectal tumor targets by acting through an A3 receptor on the effector cells. We suggest that this mechanism of immunosuppression, secondary to tissue hypoxia, may be important in the resistance of colorectal and other solid cancers to immunotherapy.

Topics: Adenocarcinoma; Adenosine; Animals; Antibodies; CD3 Complex; Cell Adhesion; Colonic Neoplasms; Killer Cells, Natural; Mice; Theobromine; Thioinosine; Tumor Cells, Cultured; Xanthines

Topics: Cell Line; Cell Survival; Colonic Neoplasms; Fluorouracil; Humans; Inosine; Thioinosine; Thymidine

In vitro resistance of HCT-8 cells to 5-fluoro-2'-deoxyuridine (FdUrd) has been obtained after a stepwise increase (up to 1 microM) in the concentration of the nucleoside in the culture medium over a period of 6 months. With a clonogenic assay, the toxicities of 17 antineoplastic agents on HCT-8-sensitive and -resistant cells were compared. Resistant cells were 700-fold resistant to FdUrd and showed different degrees of cross-resistance to several purine and pyrimidine nucleoside analogues; no cross-resistance was noted to base analogues and other cytotoxic drugs. The activities of FdUrd phosphorylase, 5'-fluorouridine kinase, 5-fluorouridine phosphorylase, 5-fluorouracil phosphoribosyltransferase, and thymidylate synthase were not significantly different in the sensitive and resistant cell lines. Mixing experiments indirectly excluded the possible elevation of the level of cytoplasmic phosphatases. The activity of FdUrd kinase in sensitive cell extracts was no more than twice that of resistant cells, and the affinities of this enzyme for FdUrd and thymidine at 0.1 to 50 microM were similar in both cell lines. However, cultures of this line failed to accumulate 5-fluoro-2'-deoxyuridylate at concentrations of FdUrd that resulted in substantial accumulation of the nucleotide in the sensitive line. These contrasting data suggested a defect in the facilitated diffusion of the analogue. The entrance of free nucleoside and its subsequent phosphorylation were compared in the two lines over short (2 to 40 s) and longer time periods at 25 degrees C and at 4 degrees C over a range of extracellular FdUrd concentrations (0.1 to 10 microM). Rapid entrance of the nucleoside into sensitive cells was observed, but entry was not detectable in resistant cells. Dipyridamole and nitrobenzylthioinosine inhibition as well as high-performance liquid chromatography analysis confirmed that data obtained from the sensitive cell line during the first 40 s primarily reflected facilitated diffusion of free nucleoside.

Topics: Adenocarcinoma; Biological Transport; Cell Line; Colonic Neoplasms; Diffusion; Drug Resistance; Floxuridine; Humans; Methotrexate; Nucleosides; Sucrose; Thioinosine