Vjera Pejanović

8-Cl-cAMP Affects Glioma Cell-Cycle Kinetics and Selectively Induces Apoptosis

8-Chloro-cyclic-adenosine-3′,5′-monophosphate (8-Cl-cAMP), a site-selective synthetic cyclic adenosine 3′,5′-monophosphate (cAMP) analog exhibits growth inhibition in a broad spectrum of human cancer lines. However, detailed studies on the effects exerted by cAMP analogs on cell-cycle kinetics have been lacking. We have examined and compared the effect of 8-Cl-cAMP on cell-cycle kinetics in two human glioma cell lines, U87MG (p53wt) and U251MG (p53mt). A flow cytometric analysis of cell-cycle distribution as well as apoptosis evaluation were performed by univariate DNA analysis after 24–72 hr of treatment with 10–50 μM concentrations of 8-Cl-cAMP. Longer incubation with 8-Cl-cAMP induced dose related accumulation of cells in S phase and a subsequent decrease in the proportion of cells in G0/G1 phase of cell cycle in both cell lines. Time-dependant suppression of cyclin B1 was detected in both glioma cell lines and could be associated with observed G2 delay. However, 8-aCl-cAMP selectively induced apoptotic cell death only in U87MG, but not in U251MG cells. Induction of apoptosis was revealed both by flow cytometry and apoptotic cell morphology. These results provide an insight into the mechanism of 8-aCl-cAMP action, suggesting that the disturbance of cell-cycle kinetics and induction of apoptosis might contribute to its growth-inhibitory effect on cancer cells.

Ribavirin reduces clinical signs and pathological changes of experimental autoimmune encephalomyelitis in Dark Agouti rats.

The effect of ribavirin on development of experimental autoimmune encephalomyelitis (EAE) was investigated. The disease was induced in genetically susceptible Dark Agouti rats with syngeneic spinal cord homogenate in complete Freunds adjuvant (SCH‐CFA). Depending on the amount of mycobacteria in CFA, the animals developed either moderate or severe EAE. Ribavirin (1‐β‐D‐ribofuranosyl‐1,2,4‐triazole‐3‐carboxamide) was applied i.p. at a daily dosage of 30 mg/kg in two treatment protocols: from the start of immunization (preventive treatment) or from the onset of the first EAE signs after the induction (therapeutic treatment). Signs of EAE began between 7 and 9 days after induction and peaked at days 11–13. In moderate EAE (mean maximal severity score 3.33 ± 0.21), the recovery was completed by days 23–26, whereas, in severe EAE (mean maximal severity score 4.5 ± 0.23), obvious recovery was not detected. Preventive ribavirin treatment significantly decreased clinical signs after both moderate (score 1.75 ± 0.25, P < 0.05) and severe (score 3.62 ± 0.31, P < 0.015) immunization. Also, disease manifestations were reduced by therapeutic treatment of ribavirin (mean maximal severity score 2.5 ± 0.2 vs. 3.33 ± 0.21 in controls, P < 0.005) but less so in comparison with preventive treatment. Analysis of the effects of ribavirin on histopathologic changes in the spinal cord tissue revealed a reduction of mononuclear cell infiltrates, composed of T cells and macrophages/microglia, and the absence of demyelination, which were pronounced in control EAE animals. Beneficial effects of preventive and therapeutic treatment with ribavirin on development of EAE suggest this nucleoside analogue as a useful candidate for therapy in multiple sclerosis.

A novel rearrangement of steroidal α-hydroxy oximes

Abstract By the action of acidic titanium trichloride upon 16-oximino-17α-benzyl-17β-hydroxy derivatives in the androstane and estrane series the 16-oxo-17β-benzyl-17α-hydroxy derivatives 6 and 7 with inversed configuration at C 17 were obtained. A mechanism for this novel rearrangement is proposed.

Sulfinosine-induced cell growth inhibition and apoptosis in human lung carcinomas in vitro

In spite of tremendous effort for improvedtherapy, lung cancer remains the leadingcause of cancer-related deaths worldwide.In the present study, we used the novelpurine ribunocleoside sulfinosine andevaluated its antiproliferative andapoptotic outcome on the non-small celllung carcinoma cell line (NSCLC) and thesmall cell lung carcinoma cell line (SCLC).Using a BrdU incorporation-test sulfinosineinhibited cell growth in a dosedependent-manner. ID50 values were4.65 ± 0.17 μM in the case of NSCLCcells, and 3.59 ± 0.81 μM in thecase of SCLC cells. MTT testing revealedthat IC50 values were 6.24 ±0.77 μM for NSCLC and 5.68 ±0.58 μM for SCLC. Inhibitoryconcentrations (IC50 and ID50)for sulfinosine were nonsignificantly lowerin SCLC cells compared to NSCLC cells,indicating similar susceptibility of thecells. Flow-cytometric analysis, TUNELstaining, DNA laddering and cell deathELISA test were used to investigateapoptotic cell death. Our resultsdemonstrated that high concentrations ofsulfinosine can cause typical DNAladdering, a hallmark for apoptosis.Evidence of free nucleosomes and enzymaticlabeling of fragmented DNA confirmedapoptosis involvement in sulfinosinecytotoxicity. In addition, flow-cytometricanalysis showed that 25 μM sulfinosinearrested cell cycle progression atthe G2M phase and induction ofapoptosis in both cell lines. From theseresults, we concluded that sulfinosine mayact as an anticancer agent and furtherstudies may prove its efficacy in lungcancer cells. Thus the biological effectsof sulfinosine may be due to modulation ofcell growth, cell death, and cell cycleregulatory molecules.

Synthesis and unusual beckmann fragmentation reaction of syn-3-Methoxy-6α,17β-Dihydroxyestra-1,3,5(10)-trien-7-one oxime

Abstract Sodium borohydride reduction of anti-3-methoxy-17β-hydroxyestra-1,3,5(10)-trien-6,7-dione 7-oxime (4a) afforded syn-3-methoxy-6α,17β-dihydroxyestra-1,3,5(10)-trien-7-one oxime (5), which in thionyl chloride at −18 °C undenvent Beckmann fragmentation reaction to the unexpected 3-methoxy-6-oxo-17β-hydroxy-6.7-secoestra-1.3.5(10)-trien-7-nitrile (6). A mechanism of this fragmentation process was proposed.

Conversion of d-xylose to protected d-lyxose derivatives and to d-lyxose, via the corresponding 1,2-anhydride

Abstract Acid hydrolysis of 3,5-di- O -benzyl-1,2- O -cyclohexylidene-α- d -xylofuranose gave the corresponding lactol, which was subsequently converted to the 3,5-di- O -benzyl-2- O -mesyl- d -xylofuranose. This compound readily reacted with sodium methoxide, sodium benzylate or sodium hydroxide (presumably via the corresponding 1,2-anhydride) to give the protected d -lyxofuranosides. These compounds were finally converted to methyl α- d -lyxopyranoside or to d -lyxose.

A nucleoside analogue, 7-thia-8-oxoguanosine stimulates proliferation of thymocytes in vitro

7-thia-8-oxoguanosine (immunosine) is a nucleoside analogue with immunoenhancing activity. In this work, its effects on proliferation of thymocytes in vitro were studied. It was found that immunosine stimulated proliferation of thymocytes both of mice and rats. The stimulatory effect depended on antigen presenting cells (APC), since thymocytes depleted of accessory cells did not proliferate to immunosine. In addition, pretreatment of APC with immunosine for 24 h significantly increased proliferation of thymocytes. Immunosine stimulated interleukin 2 (IL-2) production and the expression of activation markers (CD25 and CD71). The upregulation of CD25 (alpha subunit of IL-2R) was detected both on thymocytes and thymic dendritic cells. Proliferation of thymocytes in the presence of immunosine was predominantly mediated by IL-2 since blocking IL-2Ralpha by specific monoclonal antibodies inhibited cell proliferation by 65-85%.

A novel fragmentation-cyclization reaction of steroidal α-hydroxy oximes

Abstract By a novel “one pot” fragmentation-cyclization reaction 17β-hydroxy-17α-substituted-16-oximino derivatives in the androstane and estrane series were converted to a new type of D-homo derivative.

17β-Hydroxy-3-methoxyestra-1,3,5(10)-triene-6,7-dione 7-Oxime

X-ray structure analysis of the title compound, C 19 H 23 NO 4 , showed an unexpected anti orientation for the 6-oxo-7-oximino function. After molecular-mechanics calculations, the H atom of the 17-hydroxy moiety, which participates in a strong hydrogen bond formed in the crystalline state, changed its orientation substantially. Some change in the conformation of ring B was also observed.

The linkage of glucose to tiazofurin decreases in vitro uptake into rat glioma C6 cells.

The aim of this study was to analyse the uptake of the synthetic nucleoside tiazofurin and glucoso-linker-tiazofurin conjugate (GLTC) into rat C6 glioma cells in vitro. Results indicated that C6 cells accumulated [3H] tiazofurin slowly with time and that accumulation was reduced by the presence of unlabelled GLTC in the medium which implies that GLTC competes with tiazofurin for transport sites. Uptake of [14C] 2 deoxy-glucose into these cells was very rapid and was not affected by the presence of unlabelled GLTC. To prove the true rate of uptake, the HPLC analysis of cellular extract was performed. After the 360 min of incubation in medium that contained 0.15 mM of tiazofurin, the sum of the concentration of tiazofurin and its metabolite thiazole-adenine dinucleotide (TAD) in the cells was a total of approximately 4.8% of the amount added to each flask. After the same period of incubation in medium which contained 0.15 mM of GLTC, the sum of concentrations of conjugate, free tiazofurin and TAD represented less than 1/3 of the total concentration measured after the incubation with free tiazofurin and was further reduced in the presence of dipyridamole. Therefore, it can be concluded that GLTC shows some affinity for the nucleoside transporter, but the actual rate of uptake is low.