Antiproliferative Activity of Cytokinine Derivatives Against HCT-15 Colon and MCF-7 Breast Cancer Cells: Cell Cycle Analysis and BSA &DNA-Binding Study

Abstract

Background. N-Isopentenyladenosine (iPA) is a member of the cytokinins, a family of plant hormones that regulate plant cell growth and differentiation. iPA is present in mammalian cells in a free form, as a mononucleotide in the cytoplasm, or in a tRNA-bound form. Kinetin Riboside (KR) is devoid of cyclin-dependent kinase inhibitory activity and displayed potent antiproliferative activity against various human cancer cell lines and induced apoptosis in human myeloid leukemia cells. Earlier research has demonstrated that KR antiproliferative and apoptogenic activities are antagonized by pharmacological inhibitors of adenosine kinase (ADK), suggesting that KR bioactivation through metabolic conversion into the nucleotide form is essential for KR cytotoxicity. Aims. Apoptotic mechanism of iPA and KR led us to study in details the dosedependent cell cycle arrest and apoptogenic effect of iPA and KR on MCF-7 breast and HCT-15 colon cell lines. On the other hand, cell cycle progression or apoptosis can be affected by activation of cell cycle checkpoints in response to DNA damage. We have also studied the interaction of iPA with DNA& BSA as a complementary information related to iPA antiproliferative activity. Methods. Human breast cancer MCF-7 and HCT-15 colon cencer cells were supplied from ATCC. Growth activity of iPA and KR in vitro was evaluated by the Sulforhodamine B (SRB) assay. Apoptosis and cell cycle profile were assessed by flow cytometry. To explore the structural changes of macromoleules on addition of ligands, UV–vis absorbance spectra of macromoleules were measured at different concentrations of ligands. Results. We have calculated an IC50 of 12.2 μmol/L for iPA and IC50 of 15 μmol/L for KR against MCF-7 and same IC50 of 2.5 μmol/L for both iPA and KR against HCT-15. The cell morphology of treated cells was affected by iPA and KR treatment and loss of adhesion, rounding, cell shrinkage and detachment from the substratum. The MCF-7 cell cycle analysis by flow cytometry showed that there was a prominent increase in the amount of sub-G1/G0 phase by iPA treatment. At the concentration of 5 μM, KR led to decrease in the percentage of cells in G0/ G1 phase, which and increase percentage of cells in S and G2/M phase. Our result from structural analysis showed interaction of iPA with DNA and the binding constant value KiPA–DNA=4.4 x 10 M together with the shift of UV-vis absorbance suggest that iPA interacts at DNA surface. The BSA binding studies showed that iPA is located along the polypeptide chains with overall affinity constant of KiPABSA=4.9 x 10 M and these spectral changes were caused by compound–BSA complex formation, in which compound iPA was strongly bound at the hydrophobic positions of the BSA supported by the hydrophobic interaction. Conclusion. We concluded that the iPA and KR have cytotoxic effect on human MCF-7 and HCT-15 cells with loss of adhesion, rounding, cell shrinkage and detachment from the substratum in treated cells. Cell cycle analysis showed an indication of the inhibition of cell growth through a mechanism of apoptosis. Our result from structural analysis showed interaction of iPA with DNA suggest that iPA interacts at DNA surface and research is required in order to demonstrate that this surface-binding effect may be related to DNA damage in MCF-7 cells.