Biomedical Engineering Reference
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carcinoma that is
10-foldmore resistant to etoposide than parental HCT116 because
it has half-normal levels of topoisomerase II [37]. We also addressed the question of
the involvement of p53 in the apoptosis cascade activated either compound by using a
human ovarian carcinoma cell line pair containing wild-type and mutant p53 (A2780/
wt; A2780/mutp53).
Five drug dilutions were selected for the reference drugs camptothecin and
etoposide and the test compound BMS-250749 (0.1
>
the
IC
50
concentrations), which were chosen from their respective IC
50
values in
cytotoxic assays employing MTT as the dye to quantify viable cell number [38]. In
addition, two time points at 24 and 48 h were used to cover roughly one cycle or
slightly more that two cell cycles for each cell line tested. Figure 5.11a-c
demonstrates the efficiency that a multiple compound comparison can achieve
across cell lines, dose-dependent treatments, and a couple of time points. Further-
more, Figure 5.11a-c provides a clearer picture of the different dynamics of the
response according to the cell background and specificity of the compound. Cell
cycle profiles were tested simultaneously with apoptotic cascades evaluation
(p85PARP) ([22], data not shown). As a summary, we can say that this experi-
mental design provides the flexibility and speed to see the results at a glance on a
screening mode.
The novel quinoline derivative, TAS-103, was reported to interact with topoi-
somerase I and topoisomerase II in biomolecular assays, but the mechanism of action
in mammalian tumor cells had not been fully elucidated. The same kind of experi-
mental approach was taken for TAS-103 as for BMS-250749 above. Specific overall
results are shown in Figure 5.12. TAS-103 induced significant apoptosis in P388/Topo
I cells at levels consistent with the effects on P388 cells implying that TAS-103 was
working by a topoisomerase-I-independent pathway. TAS-103 and VP-16 (etoposide)
induced the same cell cycle arrest pattern in HCT116 cells after 24 h. HCT116/Topo II
cells, which have lower levels of topoisomerase II, needed 10 times higher con-
centration of both drugs to show the same cell cycle arrest pattern. Thus, there was no
evidence that TAS-103 was circumventing this topoisomerase II resistance effect by
acting on topoisomerase I, suggesting only a pure topoisomerase II mechanism of
action. TAS-103 induced G
1
arrest and apoptosis in p53 wt cells (A2780wtp53), but
only arrested in G
2
/M in p53 mutant (A2780mutp53). These three lines of evidence
indicate that TAS-103 activity in cells is p53 dependent and mediated by its action on
topoisomerase II and not on topoisomerase I [39].
TAS-103 induced hyperchromicity to propidium iodide after 48 h exposure; this
effect was absent in VP-16 treated cells, although the arrest pattern was maintained.
This hyperchromic effect always occurs at G
2
/M arrest, but, unfortunately, there was
no explanation for this observation. This was the only difference between TAS-103
and VP-16 and suggests the existence of an additional unknown target that may add or
synergize with the response of TAS-103/topoisomerase-II-mediated killing or may
contribute to a toxic side reaction. Either possibility will require further studies to
identify them. Overall, these studies provide helpful landmarks for the understanding
of the activities of these compounds and lower the interest in pursuing TAS-103 for
development.
,1
,5
,10
, and 20
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