aphidicolin and Carcinoma--Squamous-Cell

Recently, the three-dimensional structure of the active site of human DNA polymerase alpha (pol alpha) was proposed based on the application of molecular modeling methods and molecular dynamic simulations. The modeled structure of the enzyme was used for docking selective inhibitors (nucleotide analogs and the non-nucleoside inhibitor aphidicolin) in its active site in order to design new drugs for actinic keratosis and squamous cell carcinoma (SCC). The resulting complexes explained the geometrical and physicochemical interactions of the inhibitors with the amino acid residues involved in binding to the catalytic site, and offered insight into the experimentally derived binding data. The proposed structures were synthesized and tested in vitro for their influence on human keratinocytes and relevant tumor cell lines. Effects were compared to aphidicolin which inhibits pol alpha in a non-competitive manner, as well as to diclofenac and 5-fluorouracil, both approved for therapy of actinic keratosis. Here we describe three new nucleoside analogs inhibiting keratinocyte proliferation by inhibiting DNA synthesis and inducing apoptosis and necrosis. Thus, the combination of modeling studies and in vitro tests should allow the derivation of new drug candidates for the therapy of skin tumors, given that the agents are not relevant substrates of nucleotide transporters expressed by skin cancer cells. Kinases for nucleoside activation were detected, too, corresponding with the observed effects of nucleoside analogs.

Topics: Aphidicolin; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; DNA Polymerase I; Humans; Keratinocytes; Keratosis, Actinic; Models, Chemical; Models, Molecular; Necrosis; Nucleic Acid Synthesis Inhibitors; Nucleotide Transport Proteins; Protein Binding; Purines; Skin Neoplasms; Thymidine

Blood samples were obtained from 80 women: Twenty of these samples were from women affected by ductal infiltrating breast carcinoma, twenty from women affected by cervix uterine cancer, and forty individuals were screened for a control group. The search for chromosome instability that is known to affect individuals with cancer was performed through chromosome analysis in nontumor cells, intending to establish frequency and different types of numerical and structural aberrations. The results, in regard to spontaneous and aphidicolin induced chromosome aberrations, showed a significantly greater frequency (p < 0.001) of chromosome fragility, as well as other numerical and structural aberrations in breast cancer patients when compared to the control group. Similar results were obtained from cervix uterine cancer patients with the exception of certain numerical aberrations in which no significant differences were found. This suggests the existence of a certain degree of chromosomal instability affecting individuals with both types of cancer. The increase in fragility may play an important role in the biologic behavior and progression of cancer.

Topics: Adult; Aged; Aphidicolin; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Squamous Cell; Chromosome Aberrations; Chromosome Fragility; Chromosomes; DNA Repair; Enzyme Inhibitors; Female; Humans; Middle Aged; Nucleic Acid Synthesis Inhibitors; Uterine Cervical Neoplasms

The effect of the DNA polymerase inhibitors adenine 9-beta-arabinofuranoside (ara-A), cytosine 1-beta-arabinofuranoside (ara-C), and aphidicolin on X-radiation sensitivity was studied in a group of exponentially growing squamous cell carcinoma cell lines. The tumour cell lines varied in radiation sensitivity, with D0 (radiation sensitivity) values ranging from 1.0 to 3.9 Gy. The addition of non-toxic concentrations of ara-A 30 min before irradiation and removal 30 min after irradiation potentiated cell killing in five of eight cell lines. Four of these five responsive cell lines were relatively radioresistant lines, having D0 > 2.0 Gy. One of the cell lines was more radiosensitive (D0 = 1.4 Gy). Ara-A was also effective in potentiating killing in the radioresistant cell lines even when added 60 min after irradiation. Pre- or post-treatment with ara-A had no effect on X-ray sensitivity of the other three relatively sensitive cell lines (D0 ranging from 1.0 to 1.3 Gy). Both ara-C and aphidicolin were effective in potentiating X-ray sensitivity in JSQ-3, a relatively resistant cell line that was sensitized by ara-A treatment, but they had no effect on the X-ray sensitivity of SCC-61, a relatively radiosensitive cell line that was insensitive to ara-A effects on X-ray response. At the concentrations used, the polymerase inhibitors were equally effective in inhibiting DNA synthesis.

Topics: Aphidicolin; Carcinoma, Squamous Cell; Cell Survival; Cytarabine; DNA Repair; DNA Replication; DNA, Neoplasm; Humans; In Vitro Techniques; Nucleic Acid Synthesis Inhibitors; Tumor Cells, Cultured; Vidarabine; X-Rays

In a previous report we have characterized cisplatin (CDDP)-resistant sublines (HLac 79-DDP1 to DDP4) of the recloned squamous cell head and neck cancer (SCHNC) line HLac 79-ML revealing significant alterations of glutathione (GSH) metabolism and drug accumulation. In order to overcome CDDP-resistance in HLac 79 cells we now investigated the effect of buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, verapamil (VRP), a calcium channel blocker that has been found to modulate resistance towards a broad spectrum of antineoplastic drugs, cyclosporin A (CSA), an immunosuppressive agent probably affecting drug pharmacokinetics, and aphidicolin (APC), a fungal metabolite interfering with DNA repair through inhibition of DNA polymerase alpha, on HLac 79 CDDP-sensitivity. Using the colorimetric MTT assay, GSH depletion with BSO led to a significant decrease of the 50% inhibitory drug concentration (IC50) in all HLac 79 sublines by dose modifying factors (IC50 CDDP/IC50 BSO + CDDP) ranging from 1.8 to 3.3. VRP, CSA or APC were not effective to overcome CDDP resistance in HLac 79 cells. The potential of BSO to modulate CDDP resistance in vitro was tested in vivo in HLac 79 tumor bearing NMRI nu-nu mice subsequently. Oral administration of BSO 7 days prior and during (days -7 to 8) CDDP treatment (3 mg/kg bw i.p. days 0, 4, 8) produced a significant prolongation of mean survival time mean as compared to chemotherapy alone. This held true for both the maternal line ML in terms of chemosensitization (CDDP: mean = 40.2 +/- 15.9 days vs. CDDP + BSO: mean = 80.3 +/- 30.4 days, p less than 0.001) and the CDDP resistant subline DDP4 in terms of partially overcoming secondary drug resistance (CDDP: mean = 56.5 +/- 13.6 days vs. CDDP + BSO: mean = 72.5 +/- 15.8 days, p less than 0.001). Enhanced toxicity of combined BSO and CDDP treatment manifested by transient 10% reduction of animal mean body weight.

Topics: Animals; Antimetabolites, Antineoplastic; Aphidicolin; Buthionine Sulfoximine; Carcinoma, Squamous Cell; Cisplatin; Cyclosporins; Dose-Response Relationship, Drug; Drug Resistance; Drug Screening Assays, Antitumor; Female; Glutathione; Head and Neck Neoplasms; Humans; Methionine Sulfoximine; Mice; Mice, Nude; Tumor Cells, Cultured; Verapamil

Among common fragile sites, fra(3)(p14.2) is the most expressed either spontaneously or after treatment with aphidicolin (APC) in lymphocyte cultures. Because recurrent chromosomal abnormalities involving the short arm of chromosome 3 in tumor tissue are present in various malignancies, including lung cancer, the induction of fra(3)(p14.2) elicited by APC was investigated with the aim of detecting possible interindividual polymorphism in its expression that might be relevant to predisposition toward cancer-related events. Thirty-four patients affected with various lung cancers (14 squamous cell carcinomas, 13 adenocarcinomas, and seven small cell carcinomas) and 14 controls (patients undergoing routine routine follow-up after coronary by-pass) were included in this study. The frequency of fra(3)(p14.2) expression was not significantly different among the patients grouped either by disease or by sex and age. It was estimated that fra(3)(p14.2) accounts for about 20% of total breakage in APC-treated lymphocyte cultures from the general population.

Topics: Adenocarcinoma; Aphidicolin; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cells, Cultured; Chromosome Fragile Sites; Chromosome Fragility; Chromosomes, Human, Pair 3; Diterpenes; Humans; Karyotyping; Lung Neoplasms; Lymphocytes