Biomedical Engineering Reference
In-Depth Information
their action [40]. The evaluation of alternative tubulin targeting compounds needs to
have an extra focus on their sensitivity to be transported by these pumps; multidrug
resistance pumps include a long list of different transmembrane proteins, many of
which belong to the ABC cassette transporters (MDR, MRP, BCRP, and also a side
population described for stem cells). In addition to the transmembrane pump proteins,
tubulin mutations can also cause inactivity of these compounds [41].
Epothilones are compounds that generally lack the ability to interact with
transporter pumping proteins. Work in the field of epothilones lead to the discovery
of ixabepilone (BMS-247550) as the next generation of microtubule stabilizing
agents [42]. BMS-247550 was selected for its superior performance as compared to
paclitaxel [43]. In the flow cytometry experiments designed to evaluate this com-
pound against a panel of cell lines presenting multidrug resistance mechanisms, it was
demonstrated that this compound was active across all these resistant cell lines. This
cell line panel was assembled based upon cell cycle profiles and arrest effects obtained
for cell lines expressingMDR activities. These cell lines were assessed for expressing
the MDR phenotype by measuring expression of 4E3, as a MDR surface marker
(structural detection), as well as quantifying Rh123 transport activity, as another
characterization, to corroborate the flow cytometry results evaluating the functional
activity on the expressed MDR pump [43]. After the selection of cell lines was done,
they were also evaluated in pairs (as shown for the topoisomerase example), where the
wild-type strain and a separate strain expressing an alteration or mutation were tested
side by side. Some of the pairs used included the HCT116 and HCT116(VM)46 pair
previously shown to overexpress MDR [37]. Another cell line pair included A2780
paired in this casewith A2780Tax (a cell linewith a tubulinmutation). Included in this
series was also the Pat-7 (a mammary cell line established from a clinical sample
overexpressing the class III
-tubulin) and the H441 lung tumor for evaluation of the
ABCG2 pump [44]. Overall, these sets of cell lines demonstrated no disruption of
the activity of BMS-247550 in the presence of any of these alterations.
b
5.4.3 Farnesyltransferase Inhibitors
5.4.3.1 Approaches Utilized for Compound Evaluation Sometimes the
evaluation of compounds requires more focus on networking cascades, and this is
the case when compounds interact with cells to produce a sequence of events marked
by reduction in proliferation but with a very modest cell cycle arrest. This kind of
profile is usually accompanied by a high level of apoptosis. In this situation, the cell
cycle profile needs to be linked to a time-lapse series for sampling and a number of
parameters need to be addressed in the experimental design. We will describe here the
evaluation of BMS-214662, which was used as a model where three activities
(proliferation, arrest, and apoptosis) were linked in a way that allow to identify
the specific sequence of triggering events.
BMS-214662 is a farnesyltransferase inhibitor with high apoptosis induction
capabilities [45, 46]. In general, farnesyltransferase inhibitors are not expected to
produce any apoptosis in short periods of time, but this compound does. Because of
this property, it was necessary to devise an assay that could simultaneously evaluate
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