Indomethacin-guided cancer selective prodrug conjugate activated by histone deacetylase and tumour-associated protease

Article abstract of DOI:10.1039/c6cc04255d

An indomethacin-guided drug delivery conjugate (IGDDC) has been designed by utilizing cancer associated elevated HDAC and CTSL activities as consecutive demasking ventures for drug activation. IGDDC exhibited preferential uptake by COX-2 positive cells bo

Full text of DOI:10.1039/c6cc04255d

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DOI: 10.1039/C6CC04255D
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Indomethacin-guided cancer selective prodrug conjugate
activated by histone deacetylase and tumor-associated protease
Received 00th January 20xx,
Accepted 00th January 20xx
Joo Hee Jang,a,§ Hoyeon Lee, Amit Sharma,a,§ Sang Min Lee, Tae Hoon Lee, * Chulhun Kang *
b,§
b
b,
b,
a,
and Jong Seung Kim *
DOI: 10.1039/x0xx00000x
www.rsc.org/
An indomethacin guided drug delivery conjugate (IGDDC) has been in various biological processes.10 HDACs modulate the
designed by utilizing cancer associated elevated HDAC and CTSL chromatin structure and function by de-acetylation of the lysine
residue on amino-terminal histone trails.11 The involvement of
activities as consective demasking ventures for drug activation.
IGDDC exhibited preferential uptake by COX-2 positive cells both
in vitro and ex vivo highlighting indomethacin role in designing
new cancer-specific drug delivery frameworks.
altered HDACs expressions in cancer pathogenesis has been
intensively scrutinized and validated.12 Currently, several
HDACs are in various development stages as monotherapy and
combination therapy.13 Likewise, upregulated cysteine
cathepsins L (CTSL) has been perceived as a hallmark in
The current thrust of the cancer treatment program is to
improve the pharmacokinetics of the drug candidates. Over a
1
4
1
cancer progression and metastasis at multiple stages. On that
account, CTSL with elevated activity and their localization
past few decades, tumor-targeted delivery strategies has been
widely investigated in chemotherapy, so as to minimize adverse
effect in perturbing normal cells function compared to the
cancer cells, thereby augmenting therapeutic efficacy. The
conventional approach in this context is to utilize the targeting
1
5
exhibited the prognostic and diagnostic value clinically.
Collectively, combining HDAC and CTSL activities as a
sequential demasking tool, Ueki et al has indeed developed a
novel anticancer drug activation system with enhanced
efficacy.16
Thus, in this report, we elucidate a novel design, synthesis
and development of indomethacin-guided drug delivery
conjugate (IGDDC) for tumor treatment. IGDDC comprises of
three parts as shown in Scheme 1, incorporated intentionally to
enhance the targeting effect. The first part adopts a NSAID
ligands selective to the over-expressed receptors or transporters
on the cancer cell surface,2 further taking advantage of
3
pathological tumor microenvironment such as acidic pH,
4
reactive oxygen species (ROS) and overexpressed enzymes at
or around cancer cell to release anticancer drugs with regained
5
cytotoxicity.
(
non-steroidal anti-inflammatory drug), indomethacin, as a
Cyclooxygenase (COX) is a prostaglandin-endoperoxide
synthase (PTGS) enzyme, which plays an integral role in the
cancer targeting unit. The second part consists of drug-releasing
system via cancer abundant HDAC and CTSL activities where
a lysine moiety can be exposed after deacetylation by HDAC
and further its amide bond is selectively cleaved by CTSL to
release the drug. The third part is doxorubicin (DOX), a well-
known topoisomerase II inhibitor, for effective anti-cancer
treatment. Thereby, the IGDDC reagent will be preferentially
taken up by cancer cells, followed by stepwise activation by
endogenous HDAC and CTSL activities resulting in DOX
release with concomittent fluorescence enhancement. In this
study, the in vitro and ex vivo cancer cell targeting ability of
IGDDC is monitored by in situ fluorescence enhancement of
DOX moiety.
6
formation of prostanoids. As compared to the normal cells,
overexpressed COX-2 levels have been associated with various
cancers such as the pancreas, colon, stomach, breast, head/neck
carcinoma, or inflammatory lesions, thus indicating their vital
role in promoting tumor growth, invasiveness, angiogenesis and
7
metastasis. Indomethacin, a commercially available nonsteroid
anti-inflammatory drug (NSAID) is a non-selective COX
isoforms inhibitor (COX-1 and COX-2). Despite possessing
such poor selectivity toward COX-2 cells, its fluorescent
conjugate has been successfully utilized in tumor selective
8
imaging. Yet, to the best of our knowledge, no indomethacin-
based tumor-targeted drug delivery conjugates has been
9
The
EDC coupling of indomethacin with
compound (88% yield) followed by tert-butyl group
deprotection in TFA/DCM yielding compound (90 %). The
selective acid protection of Lys(Ac)-OH using the tert-
butylacetate/HClO (26% yield) and EDC coupling with
produced compound (60 yield). The subsequent
deprotection reaction followed by conjugation with DOX via
HATU coupling resulted in the formation of the in 24 % yield
detailed synthetic procedure in the ESI†). All compounds were
1 (IGDDC) was synthesized as shown in Scheme 1. The
attempted.
1
7
5
in DMF resulted in
The epigenetic enzyme, histone deacetylases (HDACs) has
garnered much attention in oncology owing to its crucial roles
6
2
4
2
a.Department of Chemistry, Korea University, Seoul 136-701, Korea. E-mail:
8
%
jongskim@korea.ac.kr
The School of East-West Medical Science, Kyung Hee University, Yongin, 446-701,
b.
1
Korea. E-mail: kangch@khu.ac.kr
Electronic Supplementary Information (ESI) available: Synthesis procedure, NMR,
MS, fluorescence, and cell imaging data. See DOI: 10.1039/x0xx00000x
(
1
13
well confirmed by H, C NMR spectroscopy and ESI-MS
spectrometry (see the ESI†).
‡
These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 20xx
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DOI: 10.1039/C6CC04255D
Fig. 1 Fluorescence spectra of
IX (a) b) Fluorescence spectra of
HDAC and trypsin (Red line),
1
(5 µM) treated with HDAC and trypsin
1
(5 µM) (Black line),
1
treated with
1
and HDAC (Blue line),
1 and trypsin
(
(
Green line), excitation at 499 nm in HDAC buffer with 1 % DMSO
Slit widths: ex 5 em 5)
To test the prominent role of indomethacin (a COX-selective
anti-inflammatory drug), in
fluorescence microscopic studies have been conducted towards
selected cancer cell lines. After incubating the cells with for
0 min, the fluorescence intensity of released DOX was
1 after stepwise HDAC recorded (Fig. 2). In the COX-2 positive cell lines (HeLa,
1 for its cancer-targeting ability, the
Scheme 1 Synthesis of
1
(IGDDC).
1
9
The drug release mechanism from
and CTSL action was monitored through the anticipated HepG2), the fluorescence intensity is significantly enhanced
spectral changes with ultra-visible and fluorescence microscopy. whereas it is slightly or little increased in other cell lines (Fig.
1
exhibited a broad absorption band at 499 nm and weak 2b). The fluorescence enhancement order observed as HeLa,
emission band at 590 nm (Fig. S2 in the ESI†). After adding HepG2 > HCT 116, MIA PaCa-2 > Caco-2, correspond to the
HDAC (1.25 mM) followed by trypsin (500 BAEE units/ml) increased COX-2 expression (Table S1 in the ESI†)19 as well as
1
8
(
possessing a similar protease activity to CTSL ) to the elevated HDAC and CTSL activities in the cells. The time-
solution, the fluorescence intensity was enhanced at 590 nm in dependent fluorescence change is shown in Fig. S3 in the ESI†.
time-dependent manner, mostly due to the DOX release from
All the results together support that indomethacin in exhibits
Fig. 1a). However, such fluorescence change was not observed a significant tumor targeting ability. The increased fluorescence
either by HDAC and trypsin alone (Fig. 1b). These results are intensity in HeLa and HepG2 cell lines further confirmed the
1
1
(
consistent with our anticipation in Scheme 2, the acetyl group is efficient activation of
cleaved selectively by HDAC activity in the first step and the
DOX is released by trypsin with the concomitant fluorescence
enhancement. Curiosity drove us to prove the mechanism of
1.
stepwise action of the
out a mass spectrometry analysis after addition of the HDAC
and CTSL to solution. The possesses a single molecular ion
1 towards two enzymes. Thus, we carried
1
1
peak at 1136.4 m/z. However, after addition of HDAC alone,
the mass spectrum showed a peak at 1095.649 which
corresponds to the m/z of 1-NH
addition of trypsin, it shows the peaks at 543.201 and 571.140,
corresponding to those of Dox and 9-NH , respectively (Fig.
S18-S21 in the ESI†). Further, by HPLC data, we have
confirmed that is cleaved by the cell extract to release DOX
Fig. S22 in the ESI†). Thus, from these results, we confirmed
2
, further upon the subsequent
2
1
(
that
1 would release DOX only after the stepwise action of two
enzymes, HDAC and trypsin, possibly CTSL in the cells.
Fig. 2 (a) Confocal and fluorescence microscopy images of
incubation time. Cell lines were treated with (5 μM) and incubated for
, 90 (confocal images) and 120 min (fluorescence microscopy images).
1 depend on
1
2
Confocal microscopy images were obtained from which excitation
wavelength was 488 nm (Laser power 10 %) and filter was long pass
5
05 nm and detector gain value was 850. Fluorescence microscopy
image was obtained at 460-490 nm excitation and long pass 520 nm
emission. (i) HeLa; (ii) HepG2; (iii) HCT 119; (iv) MIA PaCa-2; (v)
Caco-2. The scale bars indicate 30 μm. (b) Comparison with the
fluorescence absolute value per cell of respective cell line.
Scheme 2 Sequential DOX releasing mechanism of
1
2
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To investigate whether the release of DOX from relies on
To further confirm the indomethacin’s targeting ability,
1
View Article Online
DOI: 10.1039/C6CC04255D
competition studies were carried out (Fig. 3a). Thus, when HDAC and CTSL activities in the cells, we conducted
HepG2 was pre-treated with various concentrations of inhibition studies using
a well-known HDAC inhibitor
indomethacin up to 150 μM for 1 h, the fluorescence intensity (trichostatin A, TSA) and a CTSL inhibitor (Z-Phe-Try-CHO)
for
1 was gradually diminished (Fig. 3a) whereas the intensity and the confocal microscopic results are shown in Fig. 4. When
with HCT 116, a COX-2 negative cell line was steady under the cells were pretreated with 200 ng/ml TSA17 for 1 h, the
similar conditions. The evaluated results for both experiments intensity was decreased about 40 and 50 % for HepG2 and
are demonstrated in Fig. 3b and 3c. Together, these results HCT 116 cell lines, respectively, compared to those of the
confirmed that indomethacin moiety in
positive cancer-targeting ability.
1
displayed a COX-2 untreated cells (Fig. 4a). Likewise, treatments of the cells with
200 μM Z-Phe-Try-CHO17 resulted in diminished fluorescence
intensities by 50 and 60 %, respectively (Fig. 4b). Taken
together, these results demonstrate that the
1 possess a higher
degree of selectivity towards cancer cells with elevated HDAC
and CTSL activities over normal cells.
Fig. 5 Effect of
viability of cells, which treated with
1
on cell viability in (a) HeLa (b) Caco-2 cell lines. The
was incubated for 72 h at 37 °C,
2
% CO at different concentration. After treatment of the MTT during
1
5
3
5
Fig. 3 (a) Confocal microscopy images of
1
depend on COX-2 inhibitor;
0 min, the visible absorption of formazan crystals was measured at
70 nm. Blue bars represent the control (untreated probe) and cell
indomethacin. Cell lines were treated with various concentration of
indomethacin (μM) for 1 h at incubator. Collected media which treated
viability was normalized by the control value. Results represent the
mean (±SEM) of four independent experiments (n=4). The statistical
signification was marked as * and *** for p < 0.05 and p < 0.001
respectively, compared with the control.
before and dissolved
1 (5 μM) in it and incubated for 15 min. Control
images were untreated indomethacin. The scale bar indicates 30 μm.
Histogram of relative fluorescence intensity per cell of (b) HepG2 cells
and (c) HCT 116 depend on various concentration of indomethacin was
represented using image J program. Results represent the mean (±SEM)
of five independent experiments (n=5). The statistical signification was
marked as ** and *** for p < 0.01 and p < 0.001 respectively,
compared with the control.
To confirm the anticancer drug effect of
HeLa and Caco-2 cells (Fig. 5). exhibited significantly higher
cytotoxicity for positive cell (HeLa) with concentration
increase from 1 to 200 μM. In contrast, cytotoxicity of is not
observed for the Caca-2 cells in similar conditions. Taken
1, it was treated to
1
1
together, these results demonstrated that
enzyme positive cells.
1 selectively went to
Fig. 6 Ex-vivo fluorescence of probe
1 from xenografted mice after tail
intravenous injection. (a) Fluorescence images and (b) histogram of
relative fluorescence intensity were obtained according to comparison
with HCT 116 (cox-2 negative) and HeLa or HepG2 (cox-2 positive)
cell line. From the top White, Blue Merge image. *p < 0.05 vs. COX-2
negative tumor HCT 116.
Fig. 4 Confocal microscopy images of
1 depend on (a) HDAC inhibitor;
trichostatin A (TSA) (b) CTSL inhibitor; Z-Phe-Try-CHO. HepG2 and
HCT 116 cell lines were treated with TSA (200 ng/ml) or Z-Phe-Try-
CHO (200 μM) for 1 h at the incubator. Collected media which treated
Last but not least, in order to examine the tumor targeting
ability of
1 in live animals, we utilized a xenograft mice models
using subcutaneously injected tumor cells. The cell lines used
under this study were human colon cancer HCT 116 (COX-2
before and dissolved
1 (5 μM) in it and incubated for 15 min. Control
images were untreated TSA or Z-Phe-Try-CHO. The scale bar indicates
2
0 μm. Histogram of relative fluorescence intensity per cell of HepG2 negative), human cervical cancer HeLa (COX-2 positive) and
cells and HCT 116 was represented using image J program. Results human hepatocellular carcinoma HepG2 (COX-2 positive).
represent the mean (±SEM) of five independent experiments (n=5). The
After tail-vein injection of 1, the distribution of its fluorescence
intensity was analyzed ex vivo (Fig. 6). As shown in Fig. 5,
statistical signification was marked as ** and *** for p < 0.01 and p <
.001 respectively, compared with the control.
0
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This work was supported by CRI (No. 2009-0081566, JSK), NRF
No. 2015R1A5A1037656, CK), the Basic Science Research
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