510
log[concentration (μg/mL)]
Figure 5. Comparison of the effects of exposure for 6 days to bullatacin against MCF-7/wt vs MCF-7/Adr cells. Values are expressed as percentages of the vehicle-treated controls with each point representing the normalized average of four values and the error bars representing the standard deviation about each average. The concentration values are log dose units in μg/mL.
Figure 6 Periodic analysis (every 24 h) of the effects of serial dilution of bullatacin against MCF-7/Adr cells. Values are expressed as average absorbances (n = 6 for the vehicle treated control, n = 4 for the bullatacin treated wells), and the error bars represent the standard deviation about each average. The concentration values are log dose units in μg/mL.
tion range of 1.0 μg/mL to 1.0 × 10-4 μg/mL (Figure 5). However, using the same doses, there was the usual plateau near the IC50 value against the parental MCF-7/wt cells. At the most concentrated dose of 1.0 μg/mL, bullatacin inhibited nearly all of the growth of the MDR MCF-7/Adr cells but only 50% of the growth of the MCF-7/wt cells (Figure 5). This observation was examined further by analyzing the growth of both cell lines periodically over the 7 days of the assay (Figures 6 and 7). In the MDR MCF-7/Adr cells, bullatacin inhibited cell growth by varying amounts in a dose-dependent fashion, i.e., nearly zero cell growth at the most concentrated dose of 1.0 μg/mL vs nearly 100% cell growth at the least concentrated dose of 1.0 × 10-4 μg/mL (Figure 6). Alternatively, the cell growth of the parental MCF-7/wt cells was only inhibited by 50%, relative to the growth of the vehicle treated controls, regardless of the dose of bullatacin (Figure 7). A linear dose–response curve in the MCF-7/Adr cells after a 6-day treatment (Figure 5) reflects dose dependent cell growth when analyzed on a daily basis (Figures 4, 6, and
Figure 7. Periodic analysis (every 24 h) of the effects of serial dilution of bullatacin against MCF-7/wt cells. Values are expressed as average absorbances (n = 6 for the vehicle treated control, n = 4 for the bullatacin treated wells), and the error bars represent the standard deviation about each average. The concentration values are log dose units in μg/mL.
Figure 8. Periodic analysis (every 24 h) of the effects of 1.0μg/mL of bullatacin against MCF-7/Adr cells with refeeding of fresh media in half of the plates. Values are expressed as average absorbances (n = 6 for both control and drug treated wells), and the error bars represent the standard deviation about each average. The “R” indicates when refeeding was initiated.
7). Likewise, dose independent cell growth was confirmed in the MCF-7/wt cells both at the end of 6 days of bullatacin exposure (Figure 5) as well as over the 7 days of the assay (Figure 7).20
Both cell lines were then analyzed to determine if they were still viable after bullatacin treatment (Figures 8 and 9). A 48-h exposure to bullatacin (1.0 μg/mL) was cytotoxic to the MDR MCF-7/Adr cells since these cells would not regrow after being fed with fresh media (Figure 8). However, the MCF-7/wt cells were able to grow to a level near that of the vehicle-treated control upon being fed fresh media; thus, bullatacin was more cytotoxic than cytotoxic to these wild-type cells (Figure 9). A similar refeeding experiment, using 1.0 μg/mL of adriamycin, showed the opposite results; the MCF-7/Adr cells were completely unaffected by the antineoplastic agent, whereas the MCF-7/wt cells were no longer viable after adriamycin treatment.20
To understand better the cytotoxicity against MDR MCF-7/Adr cells and to suggest the structures optimum for future synthetic and biological evaluation, a SAR study was