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Fig. 4 Acridine orange staining of small cell lung cancer line (GLC4)
cells after exposure for 1 h to compounds 1, 4 and 6 (8 mM). (a) Untreated
cells (control). (b) Cells treated with 4. (c) Cells treated with compound 6.
(d) Cells treated with 2.
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The in vitro ionophoric activity of compounds 1–6 on small
cell lung cancer line (GLC4) cells was studied using vital
staining with acridine orange (AO). This cell permeable dye
exhibits a characteristic orange fluorescence emission in acidic
compartments such as lysosomes as a result of its protonation
and accumulation in such organelles, whereas it emits green
fluorescence at higher pH.15 When GLC4 cells were stained
with AO, the nuclei and the cytoplasm showed green fluores-
cence, while granular orange fluorescence was observed in the
cytoplasm (Fig. 4a), suggesting that the orange fluorescence is
due to acidified lysosomes. Cells were treated with 8 mM of
compounds 1–6 and the changes in the fluorescence monitored
over 1 h. Representative results are depicted in Fig. 4. Cells treated
with compounds 4 and 6, the most active carriers identified in
the liposome experiments, showed a complete disappearance
of orange emission (Fig. 4b and c). Similar results were obtained
with 1 and 5. On the other hand, cells treated with tambjamine
E 2, a poor ionophore, showed no changes (Fig. 4d). These
results correlate well with the activity observed in the liposome
assays. Active ionophores induce an increase in the lysosomal
pH whereas the inactive 2 is not able to do so. Based on the
results obtained in the liposome assays, it might be supposed
that facilitated influx of bicarbonate to the interior of the
lysosomes could be responsible for the increase of the internal
pH although other mechanisms cannot be ruled out.
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The results described demonstrate that tambjamine alkaloids
are natural anionophores and their structure is an excellent
motif to develop new anion transporters. The straightforward
synthesis of unnatural analogs of the tambjamine family
makes these compounds promising candidates to identify
new anion transporters with potential biological applications.
Efforts aimed at understanding the structure–activity relation-
ships between the ion transport and biological activity of these
compounds are now underway in our laboratories.
This paper is dedicated to Prof. Pilar Prados on the occasion
of her retirement. The authors thank financial support from
Consejerı
(project BU005B09) and the Ministerio de Ciencia e Innovacion
of Spain (projects CTQ2009-12631-BQU and Ramon y
a de Educacion de la Junta de Castilla y Leon
´ ´ ´
´
Cajal contract (R.Q.)). We also thank Marta Mansilla and
Dr Jacinto Delgado (SCAI-Universidad de Burgos) for the
X-ray determination.
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Notes and references
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c
1558 Chem. Commun., 2012, 48, 1556–1558
This journal is The Royal Society of Chemistry 2012