The Effect of Combination of CDK Inhibitor (Roscovitine) and Derivatives of Tetracycline on Human Leukemic Cells.

Roscovitine is a 2,6,9- trisubstituted aminopurine analogue that compete with ATP for binding to the active site of Cyclin-dependent kinases (CDKs). It inhibits CDK2/cyclinE, CDK7/cyclinH and CDK9/cyclinT. The cytotoxic effect of roscovitine and its analogues has been reported in several cancer cell lines in vitro and in animal models of cancer xenografts in vivo. The analogues of tetracycline (Doxycycline and Minocycline) possess anti-tumor, anti-metastatic and anti-angiogenesis properties with low toxicity besides acting as antibiotics. The effect of single agent is usually limited in cancer therapy and the use of single agent may mediate drug resistance. We have studied a cytotoxic effect of the CDK inhibitor roscovitine and tetracycline derivatives either alone or in combinations in human leukemia cells lines HL-60 (myeloid), Jurkat (lymphoblastic) and K562 (CML). HL-60, Jurkat and K562 cells were cultured in RPMI 1640 supplemented with 10% FBS. Cells were treated with roscovitine (0.5 – 100 μM), doxycyline (0.5 – 100 μg/ml) or minocycline (0.5 – 100 μg/ml) up to 48 hours alone or in combinations. The cells were examined for viability using AlarmaBlue assay, proliferation using 3H-thymidine incorporation assay, apoptosis using morphological criteria in Giemsa staining, cell cycle using propidium iodide and flow cytometry. Mitochondrial membrane permeabilization was assessed using tetramethylrhodamine ethyl ester. Specific proteins were detected by Western blotting. Incubation with roscovitine, doxycycline or minocycline decreased the cell viability in concentration-dependent manner in all three cell lines. Pretreatment of cells with doxycycline and minocycline for 24 hours followed by roscovitine for 24 hours have shown the additional decrease in cell viability. No such effect was observed when the drugs were used concomitantly. Roscovitine alone decreased proliferation in concentration- and time-dependent way in all three cell lines. Apoptotic morphology was firstly observed at 3 hrs after the treatment with roscovitine and markedly increased at 6 hrs in HL60 and Jurkat cells, but not in K562 cells. In HL60 and Jurkat cells, the cell cycle analysis has shown an increase in sub-G1 cells at 6 hours with maximum at 24h without preceding cell cycle arrest. In K562 cells sub-G1 peak increased subsequently to G2/M arrest. A marked loss of ΔΨm was observed at 2 hrs of exposure to 25uM of roscovitine in Jurkat and HL-60 cell lines, respectively. In HL60 and Jurkat cells, release of cytochrome c, AIF and Smac from mitochondria into cytosol were detected along with activation of caspases and PARP. However in K562, no caspase activation was detected at studied time points. Thus, all three agents have a cytotoxic effect in different types of leukemic cells, however, the additional cytotoxic effect of combination of Roscovitine and derivatives of tetracycline might be a potent therapeutic approach in treatment of leukemia.