Subcellular localization of a fluorescent artemisinin derivative to endoplasmic reticulum

Article abstract of DOI:10.1021/ol902890j

"Chemical eqation presented" A cytotoxic artemisinin derivative conjugated with a fluorescent dansyl moiety was synthesized and Its subcellular localization In Hep3B cells was examined. Comparison of the localization signals of the fluorescent artemisinin

Full text of DOI:10.1021/ol902890j

ORGANIC
LETTERS
2010
Vol. 12, No. 7
1420-1423
Subcellular Localization of a
Fluorescent Artemisinin Derivative to
Endoplasmic Reticulum
Yungen Liu,† Chun-Nam Lok,† Ben Chi-Bun Ko,† Tina Yuen-Ting Shum,†
Man-Kin Wong,*,‡ and Chi-Ming Che*,†
Department of Chemistry, The UniVersity of Hong Kong, Pokfulam Road, Hong Kong,
China, and Department of Applied Biology and Chemical Technology, The Hong Kong
Polytechnic UniVersity, Hung Hom, Hong Kong, China
cmche@hku.hk; bcmkwong@inet.polyu.edu.hk
Received December 15, 2009
ABSTRACT
A cytotoxic artemisinin derivative conjugated with a fluorescent dansyl moiety was synthesized and its subcellular localization in Hep3B cells
was examined. Comparison of the localization signals of the fluorescent artemisinin derivative with organelle specific dyes revealed that
endoplasmic reticulum (ER) is the main site of its accumulation.
Artemisinin (Qinghaosu, 1) is a sesquiterpene lactone en-
doperoxide isolated from an ancient Chinese herb Artemisia
annua (Sweet wormwood).1 Artemisinin and its derivatives
are highly effective against multidrug resistant malaria caused
by Plasmodium falciparum, and are currently used for clinical
treatment of malaria.2 Given the remarkable success of
artemisinin in the treatment of malaria, there is a growing
interest in exploring the anticancer activities of this class of
compounds.3 Despite hundreds of cytotoxic artemisinin
compounds known in the literature, studies on their mech-
anism of action are in the rudimentary stage. It is generally
accepted that the endoperoxide moiety is crucial to the
biological activities. Decomposition of the endoperoxide in
a reducing environment (Fe2+, GSH, etc.) leads to reactive
† University of Hong Kong.
‡ Hong Kong Polytechnic University.
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10.1021/ol902890j  2010 American Chemical Society
Published on Web 03/01/2010

recent study revealed that carbon-centered radicals generated
from activation of the endoperoxide triggered apoptosis in
cancer cell lines.5
We have previously reported a series of artemisinin
compounds that are cytotoxic to cancer cells.6 Artemisinin
derivative 2 bearing a lipophilic carbon chain (C16H33)
exhibits potent cytotoxicity against the HepG2 cell line (IC50
) 0.46 µM, Figure 1) whereas the natural artemisinin (1) is
reported the synthesis of 11-N-artemisinin dansyl amide. The
objective of this work was to prepare a fluorescent cytotoxic
artemisinin derivative and employ it in cellular localization
study. A modular approach6a of “artemisinin + linker +
lipophilic alkyl carbon chain” was used for the synthesis of
fluorescent and cytotoxic artemisinin compounds. We pro-
posed to prepare a small sized fluorescence probe with
moderate lipophilicity and chose the dansyl fluorophore
which has previously been used by Cooper8 and us9 in the
synthesis of fluorescent derivatives of natural products. The
fluorescent artemisinin compound 10 was designed. Amide
bond was selected as the linker and the C10 site of
artemisinin was linked to dansyl glycine using a carbon chain
rather than an ether group to avoid hydrolysis of the probe
under physiological conditions. Comparison of the colocal-
ization of 10 with organelle specific dyes revealed that
endoplasmic reticulum (ER) is the main subcellular site of
accumulation of the fluorescent artemisinin compound 10.
As depicted in Scheme 1, alcohol 6 was obtained from
10ꢀ-allylartemisinin (5)6a by hydroboration. p-Toluene-
Figure 1. Artemisinin (1) and its derivatives (2-4).
only weakly cytotoxic against the same cell line (IC50 ) 97
µM). By incorporation of a carboxylic acid group at the
amide linker, water-soluble derivative 3 (IC50 ) 0.18 µM,
see the Supporting Information) was prepared and found to
display cytotoxicity comparable to that of 2. The deoxy-
artemisinin derivative 4 was found to be nontoxic toward
the HepG2 cell line (Figure 1).
Scheme 1. Synthesis of Fluorescent Artemisinin 10
Comparing with the natural artemisinin (1), the strong
cytotoxicity displayed by artemisinin derivatives 2 and 3
reveals that the latter two compounds may exert cytotoxic
activities on specific cellular targets. In the literature,
fluorescent derivatives of natural products have been em-
ployed as probes to identify their subcellular localizations.
There are only several fluorescent artemisinin compounds
reported and these have been used in the antimalarial
mechanistic studies. For examples, O’Neill and Bray7
prepared artemisinin-O-acridine, artemisinin-N-acridine, and
artemisinin-N-NBD and employed these compounds to study
the antimalarial mechanism of action (see the Supporting
Information, Figure S1). Cooper and co-workers8 synthesized
artemisinin-dimer-O-coumaryl and artemisinin-dimer-O-dan-
syl compounds. Very recently, Haynes and co-workers2i
sulfonate artemisinin 7 was synthesized by coupling of
alcohol 6 with p-toluenesulfonyl chloride in 98% yield.
Nucleophilic substitution of 7 with piperazine gave 8 in 90%
yield. The fluorescent artemisinin derivative 10 was obtained
in 92% yield by coupling of amine 8 with dansyl glycine 9.
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Figure 2. (a) Electronic absorption spectrum of 10 in H2O. (b)
Emission spectrum of 10 (5 × 10-5 M) in H2O at 298 K (λex
)
331 nm).
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Org. Lett., Vol. 12, No. 7, 2010
1421

The electronic absorption and emission spectra of 10 are
depicted in Figure 2. Compound 10 in water shows absorp-
tion peak maxima at 209 (ε ) 33 500 dm3 mol-1 cm-1), 246
(ε ) 11 500 dm3 mol-1 cm-1), and 331 nm (ε ) 3 700 dm3
mol-1 cm-1). Upon excitation at 331 nm, an aqueous solution
of 10 shows an emission at 495 nm (peak maximum) with
a lifetime of 6.7 ns and a quantum yield of 0.053.
Compound 10 was found to be cytotoxic to a panel of
cancer cell lines (Table 1) after 6 days treatment at the
Table 1. Cytotoxicities of 10 against a Panel of Cell Lines
cell line HepG2 Hep3B HeLa SUNE1 MCF-7 CCD-19Lu
IC50 (µM)
8.7
7.8
10.0
1.4
3.4
22.0
micromolar level. As compound 10 is both cytotoxic and
fluorescent, it was employed as a molecular probe for
subcellular localization study in this work.
To examine the subcellular localization of 10, ER Tracker
(ER specific dye, Figure 3), BODIPY TR ceramide (Golgi
Figure 4. Colocalization of 10 with BODIPY TR ceramide (Golgi
specific dye) in Hep3B cells at different time points (1 and 24 h). Top
panel: Cellular localization of 10. Middle panel: Cellular localization of
BODIPY TR ceramide. Bottom panel: Overlay of the upper two images.
Figure 3. Colocalization of 10 with ER Tracker (ER specific dye)
in Hep3B cells at different time points (1 and 24 h). Top panel:
Cellular localization of 10. Middle panel: Cellular localization of
ER Tracker. Bottom panel: Overlay of the upper two images.
specific dye, Figure 4), and MitoTracker (mitochondria
specific dye, Figure 5) were employed for colocalization
studies. The subcellular localization of 10 in Hep3B cells
was examined. The minimum concentration of 10 for the
study was optimized at 5 µM. The cells were treated with
Figure 5. Colocalization of 10 with MitoTracker (mitochondria
specific dye) in Hep3B cells at different time points (1 and 24 h). Top
panel: Cellular localization of 10. Middle panel: Cellular localization
of MitoTracker. Bottom panel: Overlay of the upper two images.
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Org. Lett., Vol. 12, No. 7, 2010

10 (5 µM) and ER Tracker for 1 h and examined with
fluorescence microscopy. The localization of 10 was viewed
as green fluorescent spots upon excitation at 350 nm (Figure
3, top-left). The perinuclear and reticulate fluorescent image
is suggestive of location at the endoplasmic reticulum (ER).
Then, the same area was excited at 550 nm to specifically
locate the ER using ER Tracker (red, middle-left). Overlap-
ping the fluorescent images of 10 and ER Tracker showed
an extensive orange image in the same area, revealing the
colocalization of these two probes (bottom-left). It is likely
that ER is a major site at which 10 was located. Similar
results were obtained for cells subjected to incubation of 10
for 24 h (Figure 3, right column). ER has an extensive
membrane network. When using a lipid stain Nile Red to
stain the membranous structure,8 extensive colocalization of
10 with cytoplasmic membranous network resembling en-
doplasmic reticulum has been observed (see the Supporting
Information, Figure S2).
Overlapping of the fluorescent images arising from 10 and
BODIPY TR ceramide (Golgi specific dye) also showed
partial colocalization of these two fluorescent compounds
(Figure 4, bottom-left), which may be related to vesicular
connection between Golgi and ER. After 24 h, the colocal-
ization became less obvious (Figure 4, bottom-right). The
fluorescent images of 10 and MitoTracker (mitochondria
specific dye) showed little colocalization, revealing that 10
is unlikely localized at the mitochondria (Figure 5).
and contribute to the cytotoxicities.10 In particular, protein
thiols could be the targets of the reactive endoperoxide of
artemisinins.11
It has been reported that artemisinin and its derivatives
specifically target the Plasmodium falciparum PfATP6, an
ortholog of sarco/endoplasmic reticulum membrane calcium
ATPase SERCA.7a,12 Nonetheless, there are also reports
revealing that artemisinin may have nonspecific molecular
targets.8,13 Although our fluorescent artemisinin derivative
appears to target ER in cancer cells, it still remains to be
determined to what extent the antimalarial and anticancer
subcellular or molecular targets of diversified artemisinin
derivatives are in common.14
We have examined the cellular localization of 10 in the
absence or presence of a 10-fold excess of compound 1, 2,
or 3. No significant difference in both fluorescence intensity
and localization of 10 was noted. We also observed that the
cellular localization of 10 was not affected by the presence
of iron chelator desferrioxamine.7,8,14b,15 Apparently, the
subcellular localization of 10 was determined by its lipo-
philicity but not affected by the trace free iron ions present
in the cells.
In summary, a cytotoxic artemisinin compound conjugated
with a fluorescent dansyl moiety was synthesized and its
subcellular localization in Hep3B cells was investigated.
Colocalization by organelle specific dyes revealed that ER
is the main site of its accumulation.
The colocalization of 10 with organelle specific dyes
(Figures 3-5) was quantified by Pearson’s correlation
analysis (see the Supporting Information, Table S1). The
Pearson’s coefficient values (Rr) for colocalization of 10 (1-h
treatment) with ER tracker, BODIPY TR ceramide, and
MitoTracker are 0.81, 0.68, and 0.39, respectively, suggesting
that ER is the major site at which compound 10 is located.
The specialized functions of ER include translation,
folding, and transport of proteins.10 ER chaperones and
enzymes are responsible for folding, disulfide bond forma-
tion, post-translational modification of the secretary, and
integral membrane proteins. As depicted in Figure 3, the
preferential accumulation of artemisinin compounds in ER
might alter the efficiency of protein processing functions,
Acknowledgment. We are thankful for the financial
support of The University of Hong Kong and Hong Kong
Polytechnic University.
Supporting Information Available: Experimental pro-
cedures and compound characterization data. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL902890J
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