Light uncages a copper complex to induce nonapoptotic cell death

Article abstract of DOI:10.1039/c3cc38927h

Cu3G is a Cu(ii) complex of a photoactive tetradentate ligand that is cleaved upon UV irradiation to release Cu. Here we show that the cytotoxicity of Cu3G increases in response to brief UV stimulation to result in extensive cytoplasmic vacuolization that is indicative of nonapoptotic cell death.

Full text of DOI:10.1039/c3cc38927h

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Light uncages a copper complex to induce
nonapoptotic cell death†
Anupa A. Kumbhar,ab Andrew T. Franks,a Raymond J. Butcherc and
Katherine J. Franz*a
Cite this: Chem. Commun., 2013,
49, 2460
Received 13th December 2012,
Accepted 4th February 2013
DOI: 10.1039/c3cc38927h
www.rsc.org/chemcomm
Cu3G is a Cu(II) complex of a photoactive tetradentate ligand that
is cleaved upon UV irradiation to release Cu. Here we show that the
cytotoxicity of Cu3G increases in response to brief UV stimulation
to result in extensive cytoplasmic vacuolization that is indicative of
nonapoptotic cell death.
Medicinal uses of copper and copper complexes have been docu-
mented as far back as the ancient Egyptians and continue to be a
subject of intense research.1–6 The bioactivity of copper stems in
part from its requirement as a cofactor in numerous metallo-
enzymes,7 its redox activity that can induce cytotoxicity under some
conditions,2 and its predilection to displace other essential metals.8
This paradox between beneficial and toxic roles requires cells to
have elaborate systems for copper regulation.9,10 This same para-
dox also provides a rich opportunity to use small molecules to
override or intervene in copper regulation pathways as a thera-
peutic strategy, for example to overcome a localized copper imbal-
ance in neurodegenerative disease,6,11 reduce a copper overload in
Wilson’s disease,12 or hijack endogenous copper to induce oxida-
tive stress in cancer cells.13–15 In these various applications, the
coordination environment around copper plays a pivotal role in
determining the cellular uptake, localization, and ultimately the
biological activity of copper.16–18 Ideally, the biological activity
would be targeted to the disease site without affecting normal cells.
We are interested in manipulating the coordination environ-
ment of copper complexes by using external stimuli to trigger a
targeted biological activity as a consequence of altered coordina-
tion chemistry. Toward these goals, our laboratory and others
have developed a series of photocaging ligands that incorporate
Scheme 1
a photolabile nitrophenyl group in the backbone of multidentate
copper chelators.19–22 Stimulation with UV light cleaves the
ligand backbone to uncage its Cu content, as shown in
Scheme 1 for our 3rd generation chelator, 3Gcage. With an
effective dissociation constant for Cu(II) of 0.18 fM at pH 7.4,
3Gcage is our best cage to date, and preliminary in vitro investi-
gations showed that it inhibits Cu-induced hydroxyl radical
formation in the dark, whereas it promotes OHꢀ production by
300% after UV photolysis.20 In principle, such a triggered altera-
tion in coordination could be used to unleash the toxic reactivity
of an otherwise nonreactive copper complex. The current study
tested this concept in three human cancer cell lines to find that
brief UV exposure increases the cytotoxicity of Cu3Gcage.
Furthermore, treated cells showed extensive cytoplasmic vacuo-
lization, an indication of a nonapoptotic cell death pathway.
The ligand 3Gcage was synthesized by following a revised
synthetic route to improve yield (ESI†). The copper complex was
prepared by refluxing ethanolic solutions of 3Gcage with CuCl2
and purifying the blue product by column chromatography on
alumina followed by crystallization from ethanol. The crystal
structure in Fig. 1 shows Cu(II) in a distorted square pyramidal
environment provided by 3Gcage with a coordinated Cl in the
apical position.‡ The formulation of the complex is therefore
[CuCl(3Gcage)], which is abbreviated Cu3G hereafter.
a Duke University, Dept. of Chemistry, 124 Science Dr, Durham, NC 22708, USA.
E-mail: katherine.franz@duke.edu; Fax: +1 919 660 1605; Tel: +1 919 660 1541
b Dept. of Chemistry, University of Pune, Pune-411007, India.
E-mail: aak@chem.unipune.ac.in; Fax: +91 020 25691728;
Tel: +91 020 25601395, Ext. 516
c Dept. of Chemistry, Howard University, Washington, DC 20059, USA.
E-mail: rbutcher@fac.howard.edu; Fax: +1 205 806 5442; Tel: +1 202 806 6886
† Electronic supplementary information (ESI) available: Including experimental
details, spectra of Cu3G challenge experiments, additional cell viability experi-
ments, and X-ray crystallography data in cif format. CCDC 914779. For ESI and
crystallographic data in CIF or other electronic format see DOI: 10.1039/c3cc38927h
A critical challenge in the general strategy of using an external
trigger to release Cu is to ensure that Cu is retained in its complex
prior to activation. Although the thermodynamic affinity of
3GCage for Cu(II) is very strong, the reducing environment within
a cell could provide a pathway for release of Cu(I) even in the
c
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Fig. 1 ORTEP diagram of [CuCl(3Gcage)] (Cu3G) along with EtOH solvent mole-
cule, showing 50% thermal ellipsoids. Selected bond distances: Cu–N2 1.961(2),
ꢀCu–N3 2.025(2), ꢀCu–N1 2.045(2), ꢀCu–N4 2.053(2), Cu–Cl 2.5433(7) Å. Selected
bond angles: N2–Cu–N3 94.20(9), ꢀN2–Cu–N1 81.81(9), ꢀN3–Cu–N1 153.48(9),
ꢀN2–Cu–N4 169.91(9), ꢀN3–Cu–N4 79.59(9), ꢀN1–Cu–N4 100.17(9), N2–Cu–Cl
100.90(7), ꢀN3–Cu–Cl 103.65(7), ꢀN1–Cu–Cl 102.85(6), ꢀN4–Cu–Cl 88.36(7).
Fig. 3 Effect of CuCl2 or Cu3G on cell viability of (a) HL60, (b) MCF-7, and (c) HeLa
cancer cell lines. Dashed lines indicate that cells were incubated with the test
compound in the dark, while solid lines indicate that cells were treated with the test
compound for 1 h, irradiated with 350 nm UV light for 90 s, then further incubated
overnight in the dark. Where indicated in (c), cells were treated with 50 mM H2O2
added with the test compound. Cell viability was assayed by CellTitre Blue after 24 h.
Error bars represent standard deviation from experiments done in triplicate.
absence of light uncaging. In order to assess this possibility, we
measured its reduction potential by cyclic voltammetry. While no
oxidation–reduction peaks were observed between +1.0 and
ꢁ1.4 V in pH 7.4 PBS buffer, a metal-based redox feature was
observed in acetonitrile with a quasireversible peak-to-peak
separation of 110 mV and a midpoint reduction potential ðE10 =2
of ꢁ0.60 V vs. Ag/AgCl (ꢁ0.40 V vs. NHE) (Fig. 2).§
Þ
suggest that while 3Gcage does provide a robust coordination
environment for retaining Cu(II) even under reducing condi-
tions, the combination of reducing conditions and the presence
of strong Cu(I) sinks could provide a pathway for Cu removal.
The cytotoxicity of Cu3G was evaluated in MCF-7, HeLa and HL-
60 cancer cells by using CellTitre-Blue, a fluorometric assay that
measures cell viability by their efficiency to metabolize resazurin to
fluorescent resorufin. As shown in the dashed blue lines in Fig. 3,
Cu3G has little effect on cell viability after 24 h incubation in the
dark at concentrations below 100 mM, although the complex is
cytotoxic at higher concentrations in all 3 cell lines. This trend
differs from that of CuCl2 (dashed green lines Fig. 3), which was
toxic at most doses to MCF-7 and HeLa cells, but actually increased
proliferation in HL60 cells. Such a response to added copper has
been observed previously.16 The difference in cell viability between
CuCl2 and Cu3G is consistent with the hypothesis that 3Gcage
retains Cu in complexed form in cell culture.
The effect of UV light on Cu3G-treated cells was tested by
exposing treated cells to UV light centered at 350 nm in a photo-
reactor for 90 s. The short exposure time was chosen because it is
sufficient to cleave Cu3G cleanly in vitro in accord with Scheme 1.20
As shown by the solid blue lines in Fig. 3, cell viability 24 h after
photoirradiation was diminished in comparison to samples held in
the dark. In contrast, UV light did not attenuate the viability of the
CuCl2-treated cells (solid green lines, Fig. 3). Furthermore, neither
the 3Gcage ligand alone, nor the ligand after photoirradiation,
reduced cell viability at concentrations up to 200 mM (ESI†).
When viewed under the microscope, photoirradiated cells
treated with Cu3G looked rounded and swollen with extensive
cytoplasmic vacuolization (Fig. 4e), whereas cell morphology was
unchanged for untreated or 3Gcage-treated cells exposed to UV
and for cells treated with Cu3G and kept in the dark (Fig. 4a–d).
Combined, these results suggest that the cytotoxicity observed in
samples treated with Cu3G and UV light is a consequence of
This reduction potential of Cu3G matches the reported value
of ꢁ0.60 V vs. Ag/AgCl recorded in DMSO for the bisthiosemicar-
bazone complex Cu(Atsm).23 Its resistance to reduction makes
Cu(Atsm) an attractive 64Cu PET imaging agent of hypoxia, since
the intact CuII(Atsm) complex is stable and washes out of normal
cells, but can be reduced and retained under hypoxic conditions.
The similar reduction potential of Cu3G to Cu(Atsm) suggests that
Cu3G may remain intact under normal cellular conditions.
To further probe its stability, we challenged a solution of
Cu3G in PBS buffer at pH 7.4 with various reducing agents or
binding agents, including ascorbate, glutathione, cysteine,
histidine, and the strong Cu(I) chelator BCS. We found that
none of these agents on their own strip Cu from Cu3G, as
evidenced by the persistence of its characteristic d–d band at
584 nm (ESI†). However, the combination of ascorbate and BCS
did cause reductive transfer of Cu(II) from Cu3G to Cu(I) in
[Cu(BCS)2]3ꢁ (see ESI†). In contrast, Cu(Atsm) was reported to
retain Cu under similar conditions.23 These combined results
Fig. 2 Cyclic voltammogram of a 1 mM solution of Cu3G in CH3CN with 0.1 M
Et4NClO4 and a scan rate of 100 mV sꢁ1
.
c
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Cu against reductive transfer would likely improve the differential
between the stimulus-activated response vs. control. Furthermore,
compounds that respond to stimuli other than UV light for metal
release are desirable.
This work was supported in part by the U.S. National Science
Foundation (CHE-1152054). A.A.K. acknowledges the Depart-
ment of Science and Technology, Government of India, for a
BOYSCAST Fellowship (SR/BY/C-10/09) and Duke University for
providing facilities.
Notes and references
‡ Crystal data for [CuCl(3Gcage)]ꢂ2(EtOH): C25H32ClCuN5O5, Mr
=
581.55 g molꢁ1; triclinic, P1; a = 10.6787(10) Å; b = 11.3255(9) Å, c =
12.1898(12) Å; a = 113.135(9)1; b = 99.670(8)1; g = 98.548(7)1; V =
1298.4(2) Å3; Z = 2; T = 123(2) K; density (calcd) = 1.487 Mg mꢁ3; 8680
reflections measured; 5179 independent reflections (Rint = 0.0256);
R1(obs) = 0.0611, wR2(obs) = 0.1822; R1(all) = 0.0651, wR2(all) = 0.1876.
§ Abbreviations: PBS (phosphate buffered saline), BCS (bathocuproine
disulfonate), Cu3G [CuCl(3Gcage)], ROS (reactive oxygen species).
%
Fig. 4 Representative phase contrast images at 40ꢃ magnification of HeLa cells
incubated for 24 h with treatments as indicated, where UV specifies photoirra-
diation prior to incubation; (a) control, no treatment, (b) control, 90 s UV
irradiation, (c) 200 mM 3GCage ligand (no Cu) with 60 s UV irradiation, (d) 100 mM
Cu3G, (e) same as d, but with 90 s UV irradiation, (f) 50 mM Cu3G + 50 mM H2O2 + 90 s
UV irradiation prior to 18 h incubation. Images for treatment f were taken at a shorter
timepoint to show the vacuolar structures evident prior to complete cell rounding as
observed in e. Scale bar represents 10 mM.
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c
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