Figure 3.

Inhibition of Topoisomerase I activity by single or two-drug combinations treatment. Purified Topo I enzyme from calf thymus was assessed. Upper part: Lane 1, supercoiled untreated plasmid pUC19, Lane 2, plasmid + Topo I (Relaxed form), Lane 3 and 4: treatment with 122.4, 800 μg/mL and 27 μg/mL, respectively. Lane 5, 6, 7 treatment with cisplatin (80 μg/mL), etoposide (1600 μg/mL) and doxorubicin (160 μg/mL), respectively. Lower part, Lane 1, supercoiled untreated plasmid pUC19, Lane 2, plasmid + Topo I (relaxed form), Lane 3, 4 and 5: combination treatment of 122.4 (0.64 μg/mL) and cisplatin (0.54 μg/mL), etoposide (0.03 μg/mL) and doxorubicin (0.0078 μg/mL), respectively. For the combination treatment the concentrations of IC99 in Table 2 were used. We further studied whether combining 122.4 with cisplatin, etoposide, and doxorubicin will permit reducing the concentrations needed for the activation of caspase-3 by cisplatin, etoposide, and doxorubicin. Following incubation of 3 × 10⁶ cells/mL overnight with 100 μg/mL of doxorubicin/etoposide/cisplatin, five-fold increases in caspase-3 activity was observed (Figure 4). Adding 122.4 enabled to reduce the concentration of doxorubicin or etoposide or cisplatin to 50 μg/mL and still reach similar caspase-3 activation (Figure 4). In order to assure that caspase-3 is the induced enzyme, pre-incubation with caspase-3 inhibitor Ac-DEVD-CHO was performed. The results imply that compound (s) within 122.4 sensitize BS-24-1 cells in such a way that doxorubicin or etoposide or cisplatin became a better activators of caspase-3. If apoptosis is considered to be an optional mechanism to kill tumor cells, it will be worthwhile to think of combining these chemotherapeutic drugs with 122.4 content.