An Endogenous Reactive Oxygen Species (ROS)-Activated Histone Deacetylase Inhibitor Prodrug for Cancer Chemotherapy

Article abstract of DOI:10.1002/cmdc.201800367

Suberoylanilide hydroxamic acid (SAHA, vorinostat) is a potent small-molecule pan-inhibitor of histone deacetylases (HDACs) approved for treatment of cutaneous T-cell lymphoma (CTCL). However, SAHA exhibits poor selectivity for cancer cells over noncancer cells. With an aim to improving its selectivity for cancer cells, we generated a novel SAHA prodrug (SAHA-OBP) that is activated in the presence of hydrogen peroxide, a reactive oxygen species (ROS) known to be overexpressed in cancer cells. The high endogenous ROS content in cancer cells triggers rapid removal of the 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl carbonyl (OBP) cap to release active SAHA. The SAHA-OBP prodrug demonstrates selective activity against multiple cancer cell lines such as HeLa, MCF-7, MDA-MB-231, and B16-F10, while remaining benign toward noncancer cells. The downstream effects of SAHA released from SAHA-OBP in cancer cells is the induction of apoptosis. SAHA-OBP was also found to be effective on multicellular tumor spheroids (MCTS). The SAHA prodrug designed in this study undergoes rapid ROS-dependent activation and imparts much-needed selectivity to SAHA for cancer cells.

Full text of DOI:10.1002/cmdc.201800367

Accepted Article
Title: An Endogenous Reactive oxygen species (ROS)-activated HDAC
inhibitor prodrug for cancer chemotherapy
Authors: Aasheesh Srivastava, Somnath D. Bhagat, Usha Singh, and
Ram Kumar Mishra
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ChemMedChem
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An Endogenous Reactive Oxygen Species (ROS)-Activated HDAC
Inhibitor Prodrug for Cancer Chemotherapy
[
a]
[b]
[b]
[a]
Somnath D. Bhagat, Usha Singh, Ram Kumar Mishra*, and Aasheesh Srivastava*
[
[
a]
b]
Somnath D. Bhagat, Aasheesh Srivastava
Department of Chemistry
Indian Institute of Science Education and Research Bhopal
Bhopal Bypass Road, Bhopal, Madhya Pradesh, India-462066
E-mail: asri@iiserb.ac.in
Usha Singh, Ram Kumar Mishra
E-mail: rkmishra@iiserb.ac.in
Department of Biological Sciences
Indian Institute of Science Education and Research Bhopal
Bhopal Bypass Road, Bhopal, Madhya Pradesh, India-462066
Supporting information for this article is given via a link at the end of the document.
Abstract: Suberoylanilide hydroxamic acid (SAHA) is a potent small
molecule pan-inhibitor of histone deacetylases (HDACs) approved for
treatment of Cutaneous T-cell Lymphoma (CTCL). However, SAHA
exhibits poor selectivity for cancer cells over non-cancer cells.
Towards improving its selectivity for cancer cells, we exemplify a
novel SAHA prodrug (SAHA-OBP) that gets activated in the presence
inhibitors of HDAC namely vorinostat (SAHA), belinostat, and
panobinostat (Figure 1a) that are used for treating cutaneous T-
cell lymphoma (CTCL), relapsed/ refractory peripheral T-cell
lymphoma (PTCL), and multiple myeloma, respectively, contain
hydroxamic acid residue to chelate the Zn(II) present in the active
site of HDACs.[ SAHA is a pan-HDAC inhibitor, i.e., it inhibits
most isoforms of the metal-dependent HDACs non-selectively.[5,6]
As a result, it can have deleterious effects even on normal cells.
For example, it is associated with the onset of cardiotoxicity,
gastrointestinal issues and hematologic complications. Vascular
calcification is another common side effect of hydroxamic acid
containing HDAC inhibitors such as SAHA.[7] Another major
challenge with SAHA based therapy in vivo is its rapid
degradation/efflux through metabolic pathways that involve either:
a) glucuronidation through UDP-glucuronosyl-transferases
(UGTs), or b) hydrolysis of hydroxamate residue to the carboxylic
acid analogue followed by 흱-oxidation (Figure 1b), leading to
lowered therapeutic efficacy.[
4]
2 2
of H O , a reactive oxygen species (ROS) known to be overexpressed
in cancer cells. The high endogenous ROS content in cancer cells
triggers rapid removal of the OBP cap to release active SAHA. The
SAHA-OBP prodrug demonstrates selective activity against multiple
cancer cell lines such as HeLa, MCF-7, MDA-MB-231 and B16-F10,
while remaining benign to non-cancer cells. The downstream effects
of SAHA released from SAHA-OBP in cancer cells is the induction of
apoptosis. SAHA-OBP was also effective on multicellular tumor
spheroids (MCTS). Thus, the SAHA-prodrug designed here
undergoes a rapid, ROS-dependent activation and imparts the much-
needed selectivity to SAHA towards cancer cells.
8]
Introduction
Histone
deacetylases
(HDACs)
modulate the acetylation levels in histone
and non-histone proteins. HDACs
regulate gene expression by compacting
the chromatin structure. Aberrant
expression of different HDAC isoforms is
associated with a variety of pathologies
including cancer.[1] Thus, inhibition of
HDAC (HDACi) by employing small
molecules has emerged as a novel
anticancer therapy with great promise.[2]
Small molecule HDAC inhibitors often
contain hydroxamic acid, benzamide and
thiol
functionalities.[3]
Of
these,
hydroxamic acid containing molecules
are most widely explored for HDACi.
Three of the four FDA-approved
Figure 1. (a) Some FDA approved HDAC inhibitors that contain hydroxamic acid residue. (b) Inactivation
pathways of SAHA. SAHA is metabolized by glucuronidation via the UDP-glucuronosyltransferase present in
cells, and by hydrolysis followed by -oxidation.
1
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There have been continuous efforts by researchers to enhance
the therapeutic efficacy of pan HDACs inhibitors while reducing
It is known that while normal cells show very low ROS levels,[18]
cancer cells, such as those from prostate and breast, show
[
9]
[19]
their side effects. Developing acyl and carbamate derivatives of
hydroxamate-based HDAC inhibitors such as SAHA, enhances
their cell permeability and hydrolytic stability[10] while improving
their pharmacokinetic properties.[11] Hydrolysis of prodrugs both
in vitro and in vivo releases the active drug. However, these
molecules still lack ability to discriminate between cancer and
non-cancer cells. Therefore, efforts are made to achieve the
increased levels of intracellular ROS.
(H
plays a crucial role in carcinogenesis and cancer development.
The H levels in cancer cells are found to be in the range of 1-
5 mM, significantly higher than those in the normal cells (~ 0.5 µM
- 1 mM).[21,22] The increased generation of H
is linked to
Hydrogen peroxide
2
2
O ) – a prominent ROS and a marker for oxidative stress –
[20]
2
O
2
2
O
2
multiple events in cancer progression such as DNA alterations,
apoptosis, necroptosis, cell death, and tumor development etc.[
23]
release of the active form of drug in specific cells through external
stimuli.[
12–14]
In one such approach, a biorthogonal precursor of
The enhanced intracellular levels of H
an opportunity to introduce cancer cell selectivity in the prodrug
design.[24,25] Previous reports have shown that H
can efficiently
oxidize the aromatic boronate esters to phenols.[
2 2
O in cancer cells provide
SAHA was designed to trigger the release of active drug only in
presence of externally added palladium-functionalized resins.[15]
Recently, a “photo-caged” SAHA prodrug was also reported by
capping the hydroxamic acid residue of SAHA with a UV-sensitive
2
O
2
26–29]
Recently,
Kim et al. and Chang et al. illustrated the biocompatibility of a
boronic pinacol ester derived conjugate of small−molecule drug,
5´-deoxy-5-fluoro-uridine and an imaging agent such as
1-(bromomethyl)-4,5-dimethoxy-2-nitrobenzene moiety. These
photo-responsive HDAC inhibitors were delivered to
macrophages to downregulate local inflammation through HDAC
inhibition.[ The use of external stimuli for prodrug activation,
however, is practically more complicated when employing in vivo.
Hence, prodrug design that employs the inherent characteristics
of target cells as stimuli for the release of active drug are more
advantageous. The altered physiology of cancer cells compared
to their normal counterparts offers opportunity to selectively target
the former. For example, a SAHA prodrug that cleaves in
presence of glutathione (GSH) was recently reported.[17] This
prodrug showed improved cancer cell selectivity owing to the
higher intracellular GSH levels in cancer cells.
camptothecin that were able to travel throughout the body of living
16]
[30,31]
mice to reach deep cancer tissues.
Thus, in recent times, 4-
boronate benzyl moiety has been conjugated to polymers/drug
molecules to cause a H -triggered self-immolation of the
conjugate,[32,33] and release of the active drug at sites having high
local H concentrations. Quinone methide, the by-product of
2
O
2
2
O
2
this reaction, is rapidly converted to the benign 4-hydroxy benzyl
alcohol (4-HBA) in aqueous medium[27] (Scheme S1).
Based on the above reports, we chose the aromatic boronate
residue as the H O -resposive unit to create a prodrug of SAHA.
2 2
For this prodrug strategy to work, the capping moiety needs to be
sufficiently labile under physiological conditions encountered in
Figure 2. (a) The chemical derivatization of SAHA with a ROS-responsive OBP-residue to yield the SAHA-OBP prodrug. A control derivative SAHA-OBz was
also synthesized for comparison. Activation of SAHA-OBP by H occurs via a cascade of reactions that produce active SAHA along with side products such
as pinacol, boric acid and quinone methide. The latter converts to 4-hydroxybenzyl alcohol (4-HBA) in the aqueous medium. (b) UV-vis changes during
activation of SAHA-OBP with H (100 µM, 30 min, red line). The quinone methide intermediate formed by the reaction of SAHA-OBP and H produces
the broad absorption around 400 nm. (c) Overlayed HPLC traces of SAHA (blue), SAHA-OBP (green), and SAHA-OBP + H (red, 30 min after addition of
). Please see Experimental Section for details. Retention times are 5.3 min for SAHA, 10.4 min for SAHA-OBP, and 3.5 min for the quinone-methide
2
O
2
O
2 2
2 2
O
2 2
O
2 2
H O
2 2
intermediate generated from the reaction. * indicates the SAHA-OBP was treated with 100µM of H O and incubated at 37 °C for 30 min.
2
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ChemMedChem
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tumor tissues to generated the active drug, SAHA. Towards this
end, we capped the hydroxamic acid residue of SAHA with ROS
responsive moiety 4-hydroxymethyl boronic pinacol ester (OBP).
The pro-drug (SAHA-OBP, Figure 2a) thus obtained was
synthetically straight forward, and could readily release the active
drug in presence of H O . It also elicited improved selectivity
2 2
towards cancer cells and efficacious action even in multicellular
tumor spheroid (MCTS) model, as detailed below.
prodrug in aqueous medium. While the prodrug was rather stable
in serum, it underwent rapid activation in the presence of H
through the mechanism outlined in Figure 2a. In particular, the C–
B bond was rapidly cleaved in the presence of H to yield the 4-
2 2
O
2
O
2
HBA and ester of hydroxamic acid. The subsequent spontaneous
1,6-elimination generates the active drug along with the quinone
methide side product.[ The formation of quinone methide by
35]
SAHA-OBP* (i.e. SAHA-OBP activated by H
observed by UV-vis spectroscopy as peak at 395 nm (Figure
b).[36] It reaches maximum value within 19 min of adding H
to
SAHA-OBP. Mass spectrometry also confirmed the formation of
active drug in the presence of H . The parent ion for SAHA was
observed at m/z 265.15 in less than 10 min of mixing SAHA-OBP
and H . Minor peaks corresponding to 4-HBA were also
observed (Figure S3). We confirmed the stability of SAHA-OBP in
human serum and plasma with time in absence of H (Figure
S4, S5 respectively).[37] To evaluate the reactivity of SAHA-OBP
with H analytical HPLC was utilized. After incubation of SAHA-
OBP with H (100 µM), two distinct peaks were apparent in the
HPLC chromatogram (red trace labelled SAHA-OBP*, Figure 2c).
The first peak (t = 3.5 min) was identified to be the quinone-
2 2
O ) could be
2
2 2
O
Results and Discussion
2
O
2
We initiated prodrug design for SAHA in order to address the key
challenges related to SAHA that include poor selectivity/
specificity towards cancer cells and low bioavailability in cancer
cells. In this context, we worked to improve the selectivity towards
the cancer cells over normal cell by using the SAHA-OBP prodrug.
2
O
2
2
O
2
2 2
O
We chose OBP capping of SAHA in order to introduce H
sensitivity to the prodrug. In brief, the SAHA-OBP prodrug (Figure
a) was synthesized by capping the hydroxamic acid residue of
SAHA with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl
carbonyl (OBP) group. In the presence of H , SAHA-OBP was
2
2
O -
2
O
2
2
R
methide side product while the second peak (t
due to SAHA (blue trace).
R
= 5.4 min) was
2
O
2
predicted to release the active drug along with non-toxic by-
products such as pinacol, boronic acid and quinone methide. In
2 2
order to compare the OBP cap, we also synthesized a H O non-
In parallel, we investigated the HDAC inhibition activity of SAHA
and SAHA-OBP on various HDAC isoforms. We also contrasted
the activity of SAHA-OBP with the hydrolytically resistant control
compound SAHA-OBz. We chose three isoforms of recombinant
human HDACs viz. isoform 1, 2 (class-I) and 6 (class-II) to test
responsive derivative having a benzyloxy carbonyl cap (SAHA-
OBz) instead of the OBP cap.
Physicochemical properties such as the lipophilicities of SAHA
and SAHA-OBP had expected differences. The cLogP values of
0.989 ± 0.5 and 4.337 ± 0.3, for SAHA and SAHA-OBP
2 2
the activity of prodrug before and upon activation by H O . The
respectively, clearly indicated significantly increased lipophilicity
of the pro-drug.[34] This is expected to aid in its efficient uptake by
the cells. We explored the hydrolytic stability of the SAHA-OBP
results of this investigation are summarized in Figure 3a. On its
own, SAHA-OBP was inactive against various HDAC isoforms.
This is in agreement with other reports where blocking of the
Figure 3. (a) The in vitro IC50 values for synthesized compounds: SAHA and the prodrugs SAHA-OBz and SAHA-OBP. *indicates the prodrugs were
pre-treated with 100 µM of H at 37 °C for 30 min. (b) and (c) Evaluation of viability(%) of various mammalian cell lines upon treatment with SAHA-
2 2
O
OBP prodrug and SAHA, respectively, at different concentrations (n=3). The non-cancerous HEK-293T cells show high cell viability even in the
presence of 1000 nM SAHA-OBP. Statistical analysis to compare the reduction in cell viability by SAHA derivative and the performed using one-way
ANOVA test *p < 0.05 and **P < 0.01. SD is ≤ 5% of the mean.
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hydroxamic acid residue inactivates SAHA.[38] However, upon
selective cytotoxicity towards all the cells (Figure 3c). Comparing
the activity of SAHA-OBP and SAHA, we observed that SAHA-
OBP was substantially less toxic to ROS deficient HEK-293T cells
than SAHA given directly (EC50= >>1 µM and ~100 nM for SAHA-
OBP and SAHA, respectively). Moreover, SAHA-OBP was found
to be more active than SAHA in cancer cells, based on active drug
content. For example, in HeLa cells, the corresponding EC50
values were ~210 nM and ~115 nM, respectively, for SAHA-OBP
(with 50 wt% active drug) and SAHA. The non-hydrolyzing SAHA-
OBz prodrug, on the other hand, was almost inactive in both cells.
This compound showed basal toxicity (10-15%) on both types of
cells, presumably due to time dependent degradation of
compound in cytosol (Figure S7).
activation by H
inhibitor. Hence, SAHA-OBP required activation by H
2
O
2
, SAHA-OBP* releases SAHA, the active HDAC
to elicit
2
O
2
potent in vitro inhibition of HDACs. Under these conditions,
SAHA-OBP* replicated the pan-HDAC inhibitory activity exhibited
by SAHA. For cross experiments, the HDAC protein was
pretreated with H
on the activity of HDAC. However, the presence of H
2
O
2
(100 µM) to explore the influence of peroxide
had no
2
O
2
particular effect on the activity of HDACs since HDACs are non-
redox enzymes that utilize the Lewis acidity of Zn(II) present in
their catalytic site to activate the acetylated protein
substrates.[39,40] From these experiments, we concluded that
SAHA-OBP produces the HDAC inhibitor SAHA only after being
activated with H
2
O
2
.
We performed additional studies in which the non-cancerous
We compared the intracellular ROS levels in cancer cell line and
normal cell line by using 2,7-dichlorofluorescein diacetate (DCF-
DA).[41] The non-fluorescent DCF-DA reagent converts to the
fluorescent DCF in presence of intracellular ROS. Indeed, in the
DCF-DA assay we observed significantly increased fluorescence
signal only in HeLa cells and not in HEK-293T cells (Figure
S6).[42,43] The HeLa cells showed 6 fold higher fluorescent
intensity than HEK-293T cells. In HEK-293T cells, the
fluorescence intensity of DCF-DA was similar to that in PBS. The
HEK-293T cells were pretreated with H
2 2
O (100 µM) for 30 min.
The H pre-treatment introduced ROS extraneously to the
2
O
2
HEK-293T cells and resulted in significant enhancement of
toxicity of SAHA-OBP fed to these cells later. For example, at 200
nM concentration of SAHA-OBP (100 nM active SAHA
concentration) 85% toxicity occurred upon H O pretreatment
2 2
while only ~ 25% toxicity was observed in the absence of the
pretreatment. These results indicate that the cytotoxicity of
2 2
prodrug derives from the H O -stimulated release of SAHA.
2 2
contrasting intracellular H O concentrations
in normal and cancerous cells should impart
SAHA-OBP with the ability to discriminate
between them. To test this, the cytotoxicity
profiles of SAHA, prodrug SAHA-OBP, and
the non-hydrolysable compound SAHA-OBz
were evaluated on HEK-293T (non-
cancerous and ROS deficient) as well as on
HeLa (cervical carcinoma), MCF-7 (breast
cancer), MDA-MB-231 (breast cancer) and
B16-F10 (musculus skin melanoma) (Figure
S7). Cancer cells of these types are known to
have a ROS overexpression.[23,44] The cells
were treated (for 24 h) with SAHA derivatives
at different concentrations (20 to 1000 nM). It
was observed that, the prodrug SAHA-OBP
did not show significant cytotoxicity to normal
HEK-293T cells even at the maximum
concentration tested (~95% viability at 1000
nM SAHA-OBP). In other words, it had half
maximal effective concentration (EC50
>1000 nM for HEK-293T cells, (black line,
Figure 3b). However, at similar
)
>
concentrations, the SAHA-OBP prodrug
displayed a very strong and dose-dependent
cytotoxicity towards B16-F10 cells (EC50~390
nM), HeLa cells (EC50~ 370 nM), MCF-7
(
EC50~340 nM) cells. SAHA-OBP was
somewhat less effective on MDA-MB-231
cells with EC50 > 1000 nM concentration since
these cells are known to have low intracellular
concentration.[
45]
Here, it must be
H
2
O
2
mentioned that the active drug content of
SAHA-OBP is only 50 wt%. Thus, a 500 nM
solution of SAHA-OBP can result in upto 250
nM active SAHA concentration upon
complete activation. In contrast to SAHA-
OBP, SAHA showed the expected non-
Figure 4. (a-d) Bright field and fluorescence images of HEK-293T cells (top row) and HeLa cells
bottom row) upon treatment with SAHA-OBP (200 nM, 6 h). Scale bar = 20 µM. (e) Comparative
cell cycle distribution profiles of HEK-293T and HeLa cells before and after treatment with SAHA-
OBP and SAHA (200 nM, 6 h).
(
4
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Figure 5. The effect of SAHA-OBP on HeLa multicellular tumor spheroids (MCTS). (a) Representative optical micrographs of control
(
(
PBS, pH = 7.2) and SAHA-OBP (active drug concentration = 200 nM) treated spheroids at different time intervals. Scale bar = 20 µm.
b) Change in the area of the HeLa MCTS without (black line, PBS treatment) and upon treatment with active 200 nM concentration
each of SAHA (blue line) and SAHA-OBP (orange line) respectively. Green arrows indicate that the dosage of SAHA-OBP and SAHA
were given for three consecutive days. Statistical analysis to compare the reduction in MCTS by SAHA and SAHA-OBP and the
performed using one-way ANOVA test *p < 0.05, **p < 0.01. SD is ≤ 5% of the mean.
Hence, we can conclude that SAHA-OBP exploits the high
intracellular ROS levels in cancer cells to elicit selective toxicity
against them.
control had only 38% in this phase (Figure 4e, S9a and S9b). On
the other hand, the non-cancerous HEK-293T cells demonstrated
very similar cell cycle distribution profiles for the untreated and the
SAHA-OBP treated cells; while treatment with SAHA showed the
cell cycle arrest in G1/S phase for these cells. Thus, SAHA-OBP
selectively induced G1-phase cell cycle arrest in the cancer cells.
Exposure of HEK-293T and HeLa cells to SAHA-OBP resulted in
time-dependent changes in the extent of -tubulin acetylation
levels. The total levels of acetylated tubulin in the HEK-293T cells
remained unaffected upon treatment with SAHA-OBP (Figure S8,
Left panel); while it enhances in HeLa cells (Figure S8, middle
panel). In the case of HeLa cells, SAHA-OBP treatment led to
progressive increase in the levels of intracellular acetylated α-
tubulin. SAHA (200 nM) treatment was used as a positive control
for the Western blotting studies, and expected enhancement of
tubulin acetylation levels was observed. The pre-treatment of
The above observations were corroborated by propidium iodide
(
2
PI) uptake assay. Upon treatment with 200 nM SAHA-OBP, HEK-
93T cells maintained good confluence and did not elicit
significant PI uptake upon treatment of SAHA-OBP (Figure 4a-b).
Contrastingly, similar exposure of HeLa cells to SAHA-OBP
significantly reduced their confluence and induced apoptosis in
them. The latter was confirmed through the propidium iodide (PI)
uptake by the treated HeLa cells (Figure 4c-d). SAHA is a pan-
HDAC inhibitor that also inhibits the cytosolic HDAC6 isoform.
Tubulin is amongst the known non-histone protein substrate for
HDAC6. Inhibition of HDAC6 enhances acetylation of tubulin. The
downstream effects of this include increased assembly of
polymerized tubulin and induction of apoptosis.[46] Hence, the
effect of SAHA-OBP treatment on -tubulin acetylation levels was
studied by western blot technique (Figure S8).
2 2
HEK-293T cells with H O resulted in SAHA-OBP activation and
consequent cytotoxicity in these cells too (Figure S7a).
We subjected HEK-293T and HeLa cells to flow cytometry
analysis to assess if there is any cell cycle arrest upon treatment
of cells with SAHA-OBP and SAHA. Treatment of HeLa cells with
In recent decade, multicellular tumors spheroids (MCTS) have
been employed as resilient three-dimensional (3D) intermediate
platforms to screen therapeutic potential of small molecules in
tumor chemotherapy.[47,48] We analyzed the anticancer potential
of SAHA and SAHA-OBP (active drug concentration = 200 nM for
200 nM SAHA-OBP and SAHA for 12 h resulted in 64% and 57%
cells, respectively, present in the G1-phase, while the untreated
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stability in human plasma. The
prodrug SAHA-OBP also exhibited
enhanced cLogP values that will
be beneficial towards improving its
cellular
exhibited long-term stability in
-deficient medium but rapidly
transformed into the active drug
through set of sequential
reactions in presence of H . The
active drug, released in presence
2 2
of high endogenous H O
uptake.
SAHA-OBP
2 2
H O
a
2
O
2
concentrations of cancer cells,
inhibited the cytosolic HDAC6
resulting in hyper-acetylation of α-
tubulin and its incorporation into
the microtubule bundles within the
treated cells. PI staining confirmed
apoptosis in the treated cells.
These findings are summarized in
Figure 6. Extraneous introduction
2 2
of H O in non-cancerous (HEK-
293T) cells could also activate the
prodrug and induce toxicity in
them. The anticancer activity of
SAHA-OBP prodrug was also
confirmed in a 3D tumor spheroid
model. We anticipate that the
hybrid prodrug SAHA-OBP will
contribute to the development of
intelligent therapeutics based on
the design of chemo-responsive
bioactive molecules that can
Figure 6: Schematic representation of the key findings of this work. The SAHA-OBP prodrug is activated
inside cancer cells due to the high intracellular ROS levels in these cells. The reaction between SAHA-
OBP and H O produces the active SAHA along with the quinone methide intermediate. SAHA inactivates
2 2
the cytosolic HDAC6, causes hyperacetylation of tubulin, and ultimately results in apoptosis of cancer cell.
The quinone methide intermediate is converted to 4-hydroxy benzyl alcohol. SAHA-OBP activation also
produces pinacol and boric acid as the other benign side-products.
potentially
provide
improved
each) on HeLa MCTS.[48] The size of HeLa MCTS was analyzed
cancer treatment. Such an approach is expected to offer a safer,
more specific and more selective activity against cancer cells.
after treatment of SAHA-OBP and control (PBS 7.2). The
treatment regimen begun on the fourth day of seeding and
continued once a day for next three days. The size of tumor
spheroids was measured from 5th day onwards and continued till
the 21st day. Representative optical micrographs at different time
points are shown in Figure 5a. It can be seen that while the control
Experimental Section
2 2
HPLC analysis of H O mediated reactions:
2
HeLa MCTS grew to about 300 mm in the course of 21 days, the
2
treated MCTS grew to only about 100 mm in this time (Figure 5b).
The HPLC chromatograms of SAHA-OBP, SAHA, and the reaction
products of SAHA-OBP and H were performed on a system with a C18
column (250 mm × 4.6 mm, 5 µm) and the conditions were as follows:
ACN/H O = 40/60 (v/v); flow rate: 0.6 mL/min; detection wavelength: 260
nm. Both samples (SAHA-OBP and SAHA) was prepared in HPLC grade
acetonitrile solvent (1 mg/ml) and 20 µl were manually injected in HPLC.
Moreover, we noted that SAHA-OBP was even more effective
than SAHA in reducing the tumor size. At 21st day, while SAHA
had achieved about ~50% reduction in the tumor size, SAHA-
OBP had achieved ~80% reduction. We attribute this to the
efficient uptake of SAHA-OBP due to its increased lipophilicity
2 2
O
2
(
vide supra). As a result, SAHA-OBP exhibits an overwhelmingly
The experiment was carried out by using reversed-phase HPLC using a
strengthened the inhibitory effect by enhancing the cell death and
growth reduction in the HeLa MCTS.
25 cm × 4.6 mm, internal diameter (i.d.) glass capillary packed with 5 µm
C18 Zorbax beads column. The SAHA-OBP and SAHA were eluted from
the C18 column into the HPLC using a linear gradient (40:60)% of ACN:
Water at a flow rate of 0.6 mL/min for 30 min. The buffers used to create
the ACN gradient are buffer A (0.2% TFA) in water.
Conclusions
Animal Cell Culture:
In conclusion, we have designed a prodrug platform that exploits
2 2
the high intra-tumoral H O levels to achieve the uncaging of the
The mammalian cell lines (HEK-293T, HeLa, MDA-MB-231, MCF-7, B16-
F10) were cultured in complete DMEM media (DMEM supplemented with
prodrug. This releases the active drug SAHA that is involved in
the epigenetic regulation of cancer. This prodrug design
addresses some of the key challenges related to SAHA that
include poor selectivity/ specificity towards cancer cells and poor
10% Fetal Bovine Serum, FBS supplemented with Penicillin and
Streptomycin under the 5% CO @ 37 °C). The cells growth was continued
2
6
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10.1002/cmdc.201800367
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2
in sterile T-flask or multi-well culture plate at 37°C in CO incubator
maintaining 5% CO and 95% humidity.
2
AS thank SERB, India for funding this work through grant no.
EMR/2016/006545. SDB and US thank UGC, India for Senior
Research Fellowships. SB acknowledges useful scientific discussions
with Dr. Abhishek Chanchal.
MTT Assay:
The MDA-MB-231, HeLa, MCF-7, B16-F10, HEK-293T cell lines were
grown in DMEM medium supplemented with 115 units/mL of penicillin G,
Keywords: Vorinostat • HDACi delivery • Prodrug • ROS •
115 μg/mL of streptomycin, and 10% fetal bovine serum (FBS). The cell
Tumor spheroids
lines HEK-293T, HeLa, MCF-7, MDA-MB-231, B16-F10 were seeded
equally at 1 x 103 cells per well in a 96-well plates in complete DMEM +
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concentrations of the compounds (20-1000 nM) shown in Figure S5. For
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dimethylthiazol-2-yl]-2,5-diphenyltratrazolium bromide (MTT reagent
prepared in 20mM PBS 7.2) and after four hours, all of the medium
including MTT solution (5 mg/mL) was aspirated from the wells. The
remaining formazan crystals were dissolved in 150 µl of DMSO and the
absorbance was measured at 540 nm using microplate reader (Synergy-
HT, Bio-Tech Instruments, Inc.). The cytotoxicity index was determined
using the untreated cells as negative control. The percentage of
cytotoxicity was calculated using the background-corrected absorbance.
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Funding Sources:
This work was financially supported by intramural funds from IISER
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UGC India for Senior Research Fellowship.
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ChemMedChem
10.1002/cmdc.201800367
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Entry for the Table of Contents
Insert graphic for Table of Contents here.
Prodrugs that can be selectively triggered by increased endogenous H
2
O
2
levels in cancer cells can impart the much needed selectivity
to cancer chemotherapy. A prodrug of Vorinostat (SAHA-OBP) is reported that shows selective and potent killing of cancer cells over
the normal cells.
9
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