This search yielded sangi vamycin as the primary homology hit along with several other analogs of this agent. Further reduction in stringency to 80% expanded the compound list to include several characterized anti neoplastic adeno sine analogs. including tubercidin, tricirib ine and toyocamycin. Based molecular weight calculator on results showing high structural similarity, both sangivamycin and toyocamycin were selected for side by side biological evaluation with ARC. As controls, the structurally distinct adenosine analog fludarabine was added along with an unrelated pyrimi dine anti neoplastic 6 thioguanine. Structures for the compounds utilized in this study are shown in Fig. 1. Cytotoxicity, apoptosis and cell cycle analysis ARC was evaluated in the context of 14C leucine viability assays using two cell lines, HL60 and MCF7.
In both lines, ARC was shown to have activity Inhibitors,Modulators,Libraries in the nanomolar range. The maximal effect occurred within the first 24 h of incubation for HL60 cells, while for MCF7 cells, the maximal effect did not occur until 3 days of incubation. Expanding this study to a panel of diverse tumor lines confirmed Inhibitors,Modulators,Libraries nanomolar activ ity in all cell lines with a 20fold variance at day 1 which declined to 11fold by day 6 showing that relative cell line susceptibility gradually disappears with prolonged incubation. Follow ing this, HL60 and MCF7 cells were treated with all 5 com pounds and IC50 values determined over the same extended time course. Results dem onstrated that in both lines, the time course of cytotoxicity was similar between ARC, sangivamycin and toyocamy cin, and markedly different from fludarabine and thiogua nine.
Having highlighted general similarities in IC50 for ARC, sangivamycin and toyocamycin in HL60 and MCF7 cells, the study was then extended to a panel of cell lines. Calculation of correlation coefficients showed high similarity in activity between ARC and Inhibitors,Modulators,Libraries sangivamycin, but not between ARC and toyocamycin. These experi ments suggest for the 5 compounds, ARC and sangivamy cin are the closest relatives in terms of growth inhibitory effects. Following this, the relative ability of ARC to induce apop tosis or perturb the cell cycle was evaluated in HL60 cells. Results from cell cycle analysis showed marked increases in the percentage of cells in S and G2M phase after treatment with ARC, Inhibitors,Modulators,Libraries sangivamycin or toyocamycin, indicating progress towards a G2M block, whereas fludara bine demonstrated a partial block in G1 and thioguanine had little effect.
Doxorubicin and cisplatin Inhibitors,Modulators,Libraries treated cells were 17-AAG buy included as controls and showed the expected S G2/ M and S phase blocks, respectively. In the context of apop tosis induction ARC, sangivamycin and toyocamycin produced identical profiles with a small increase in levels of apoptosis after 48 hr treatment. Thus ARC, sangivamycin and toyocamycin behave similarly in these cell based assays.