Bis-arylidene oxindole-betulinic acid conjugate: A fluorescent cancer cell detector with potent anticancer activity

Article abstract of DOI:10.1021/acsmedchemlett.5b00095

Molecules offering simultaneous detection and killing of cancer cells are advantageous. Hybrid of cancer cell-selective, ROS generator betulinic acid and bis-arylidene oxindole with amino propyl-linker is developed. With intrinsic fluorescence, the molecu

Full text of DOI:10.1021/acsmedchemlett.5b00095

Letter
pubs.acs.org/acsmedchemlett
Bis-Arylidene Oxindole−Betulinic Acid Conjugate: A Fluorescent
Cancer Cell Detector with Potent Anticancer Activity
Abhishek Pal,†,§,∥ Anirban Ganguly,‡,∥ Sumit Chowdhuri,† Md Yousuf,† Avijit Ghosh,†
Ayan Kumar Barui,‡ Rajesh Kotcherlakota,‡ Susanta Adhikari,*,† and Rajkumar Banerjee*,‡
†Department of Chemistry, University of Calcutta, 92, A.P.C. Road, Kolkata 700 009, India
‡Biomaterials Group, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
§Department of Organic Chemistry, Indian Association for Cultivation of Science, Jadavpur, Kolkata 700 032, India
S
* Supporting Information
ABSTRACT: Molecules offering simultaneous detection and killing of cancer cells
are advantageous. Hybrid of cancer cell-selective, ROS generator betulinic acid and
bis-arylidene oxindole with amino propyl-linker is developed. With intrinsic
fluorescence, the molecule exhibited cancer cell-specific residence. Further, it
generated ROS, triggered apoptosis, and exhibited potent cytotoxicity in cancer
cells selectively. We demonstrate the first example and use of isatins as betulinic acid
conjugate for selective detection of cancer and subsequent killing of cancer cells via
apoptosis.
KEYWORDS: Fluorescent oxindole, betulinic acid, cancer cell detection, ROS, apoptosis
ancer treatment needs multimodal strategies to restrain its
resistance against BetA-mediated toxicity, favorable therapeutic
Caggressive malignancy. Strategies that include simulta- window is naturally expected. Evasion of apoptosis is one of the
hallmarks of cancer.18 Therefore, BetA with natural tendency to
neous detection/sensing and treatment should be specific for
trigger mitochondrial pathway of apoptosis may find use in
treating drug resistant cancers.16 BetA is hence used in various
combination strategies including chemotherapeutic drugs,19,20
death receptor ligand TRAIL,21 etc. BetA is a known reactive
oxygen species (ROS) scavenger for normal cells22,23 but
generates excessive ROS to promote a caspase-independent
programmed cell death in cancer cells.17,24 Cancer cells
generate moderate level of ROS, which accounts for DNA
damage promoting enhanced genetic instability and drug
resistance.25 However, the excessive increase in ROS-mediated
oxidative stress misbalances cancer cells and overwhelms
antioxidant-defense of endogenous reduced glutathion (GSH)
and super oxide dismutase (SOD). Hence, selective and
excessive ROS generation is a strategically useful anticancer
tool.26,27 However, the entry of BetA in cancer cells is not
selective as there is evidence for its entering in nonproliferating
normal cells, wherein it acts as ROS-scavenger. However, ROS-
scavenging or ROS-generating role of BetA in proliferating
normal cells (such as in blood, skin, or hair cells) is not clear.
Implicitly, in order to avoid any unwanted toxicity BetA should
be properly targeted.
cancer to avoid unwanted side effects. Effective detection
includes specific labeling of tumor cells. Desirably, detection,
drug-sensitization, and effective cytotoxicity among tumor cell
population and various infiltrating cells in tumor-microenviron-
ment would be the most sought-after strategy for controlling
tumor-aggressiveness. Among tumor-detecting molecular
probes, intrinsically fluorescent small molecules are preferably
and widely employed as they can enter live cells easily and offer
screening through visual detection.1−5
Isatin-based molecules are biologically important, but there
are limited reports toward establishing isatin-based fluorescent
compounds for biological screening.6−8 To our knowledge, use
of fluorescent isatins in the context of cancer detection is not
documented before. This is probably because isatins so far
developed did not show any specificity toward cancer cells.
Recently we reported a new class of antiestrogenic, bis-arylidene
oxindoles (certain derivatives of isatin) as antibreast cancer
agent.9 In our pursuit for designing a cancer cell selective
molecular probe, we have focused our attention toward these
intrinsically fluorescent-isatin molecules.
Natural triterpenoid betulinic acid (BetA) exhibits a range of
biological effects including potent antitumor activity.10−16 This
anticancer property is linked to its ability to induce caspase-
independent apoptotic cell death in cancer cells by depolarizing
mitochondrial membrane potential.17 Since normal cells exhibit
Received: March 1, 2015
Accepted: April 13, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00095
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Toward developing a new hybrid molecule strategically
exhibiting cancer cell selective detection and killing, we
synthesized bis-arylidene oxindole-conjugated BetA. Since
these isatins exhibit fluorescence properties, we reasoned that
upon conjugation we could visualize the fate of BetA conjugate
in cells. The synthetic procedure for the hybrid molecule is
illustrated in Scheme 1. The precursor bis-phenol derivative 3
of BetA-FITC was accomplished as per the described synthetic
procedure (Figure S2).
To determine if any slight structural change would affect the
cellular uptake, we opted to test Is-PMB and Is-amine
compounds along with Is-BetA for their uptake properties in
various cells. As absorptivity of tested molecules emitting at red
region were not the same, therefore, for comparison of
quantitative cellular uptake of each molecule, we represented
data as % uptake following normalization with fluorescent
absorption values of respective molecules. As a positive control
we used anticancer drug, doxorubicin (Dox), which also
possesses red fluorescence. One noncancer cell, NIH 3T3,
and two cancer cells, B16F10 (mouse melanoma) and PANC-1
(human pancreatic cancer), were used for this uptake study.
The microscopic visualization of cells (Figure 1A) treated with
Scheme 1. Synthesis of bis-Arylidene Oxindole−Betulinic
a
Acid Conjugate (Is-BetA, 7)
a
Reagents and conditions: (i) (a) NaH, THF, r.t., 1 h; (b) PMB-Br,
DMF, r.t., 5 h, 78%; (ii) 4,4′-dihydroxy benzophenone, Zn, TiCl4,
THF, reflux, 3 h, 65%; (iii) (a) K2CO3, acetone, reflux, 1 h; (b) 1,3-
dibromo propane, reflux, 3 h, 60%; (iv) NaN3, DMSO, r.t., overnight,
98%; (v) (a) PPh3, THF, rt, 2 h; (b) H2O, r.t., overnight 75%; (vi)
betulinic acid, DCC, DMAP, HOBT, DMF, r.t., overnight, 85%.
(Is-PMB) was synthesized using two-step reaction sequences
starting from isatin following our published protocol.9 Mono
O-alkylation of compound 3 using 1,3-dibromo propane and
potassium carbonate as a base in acetone gave compound 4 in
60% yield. As our expectation, compound 4 was obtained as an
inseparable mixture of Z and E isomers. Nucleophilic
substitution of this bromo-compound (4) using sodium azide
in DMSO yielded compound 5 in 98% yield. Staudinger
reduction of compound 5 in the presence of triphenylphos-
phine in THF provided compound 6 (Is-amine) in 75% yield.
Finally compound 7 (Is-BetA) was synthesized by reacting
compound 6 with BetA using N,N′-dicyclohexylcarbodimide
(DCC), 4-dimethylaminopyridine (DMAP), and hydroxyben-
zotriazole (HOBt) in DMF with 85% yield.
All the three synthesized compounds and intermediates, Is-
BetA (compound 7), Is-PMB (compound 3), and Is-amine
(compound 6) incidentally showed red fluorescent properties,
with their excitation λmax ranged within 369−383 nm, and
emission λmax, 552−565 nm, respectively (Figure S1). Betulinic
acid (or BetA) does not possess any fluorescent property. In
order to visualize the extent of cellular entry of BetA, which is
originally nonfluorescent, we conjugated BetA with green
fluorescent fluorescein molecule to form BetA-FITC. The
fluorescent modification of BetA was done similar to that of the
synthesis of Is-BetA, wherein carboxylic acid group of BetA was
involved. We hypothesized that BetA-FITC and Is-BetA would
not largely differ in their extent of cellular entry as the
conjugation pattern (involving BetA) did not differ. Synthesis
Figure 1. (A) Fluorescence images of normal cells (NIH 3T3) or
cancer cells (B16F10, PANC1) either kept untreated (UT) or treated
with Is-PMB, Is-amine, BetA-FITC, Is-BetA, and Dox, respectively.
(B) Quantitative uptake of Is-BetA, Dox, Is-PMB, Is-amine, and Bet-
FITC in cells as depicted. The data is represented as percentage of
fluorescent molecules’ uptake per microgram of protein.
various molecules revealed the following: (a) noncancer
(NIH3T3) cells did not take up Is-BetA, Is-amine, and BetA-
FITC, but took up Is-PMB and Dox quite efficiently. (b)
Contrastingly, cancer cells took up all treated molecules
including Is-BetA, Is-amine, and BetA-FITC. Quantitative
analysis of this uptake study maintains this microscopically
visualized trend (Figure 1B). In consonant with microscopic
observation, Is-PMB and Dox showed significantly higher
percentage of uptake in both cancer and noncancer cells
nonspecifically. Interestingly, Is-amine and Is-PMB, which bear
minimum structural difference, exhibited contrasting uptake
behaviors. It is the presence of O-propyl amine side chain
spacer in Is-amine that possibly bolstered its (and also Is-
BetA’s) selective uptake in cancer cells. Moreover, BetA-FITC
individually exhibited selective uptake in cancer cells. We
believe this might have synergized the cellular uptake of Is-BetA
conjugate in cancer cells. Detailed microscopic images are
provided in Figures S3−S5.
B
DOI: 10.1021/acsmedchemlett.5b00095
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Table 1. Cytotoxicity of Is-BetA and Other Derivatives in Cancer (MCF-7, B16-F10, OVCAR 3, MDA-MB-231, PANC-1, and
A549) and Noncancer Cells (CHO, NIH3T3) Following 72 h of Continuous Treatment (ND, Not Determined)
MCF7 (IC50,
B16-F10 (IC50, OVCAR 3 (IC50,
MDA-MB-231
(IC50, μM)
PANC1 (IC50,
A549 (IC50,
CHO (IC50, NIH 3T3 (IC50,
μM)
μM)
μM)
μM)
μM)
μM)
μM)
Is-BetA
BetA
7.396 0.034
10.527 0.024
ND
2.296 0.037
6.048 0.018
8.366 0.008
ND
1.256 0.004
2.630 0.032
10.223 0.022
9.778 0.033
2.560 0.022
>20
>20
ND
>20
>20
>20
>20
20
>20
ND
>20
ND
BetA-
FITC
Is-amine
Is-PMB
Dox
ND
>20
ND
>20
ND
>20
ND
>20
>20
ND
>20
1.359 0.004
ND
>20
ND
ND
>20
>20
>20
ND
ND
0.663 0.024
1.026 0.036
7.211 0.029
The conjugate molecule Is-BetA, its precursors such as Is-
Amine and Is-PMB, and its conjugate partner BetA were tested
in several cancer and noncancer cells for their cytotoxic effect.
Dox was also included in this study. This study was done with
following cancer cells: MCF-7 (human breast cancer), A549
(human lung cancer), PANC-1 (human pancreatic cancer),
B16F10 (mouse melanoma), OVCAR-3 (human ovarian
cancer), and MDA-MB-231 (human breast cancer). The
noncancer cells as included for this study were CHO (chinese
hamster ovary) and NIH3T3 (human fibroblast). The result is
presented in Table 1. Even though Is-PMB showed nonspecific
cellular uptake in both cancer and noncancer cells, it did not
show any toxicity in these cells. Dox was as usual toxic in both
kinds of cells. BetA and Is-amine, which were selectively taken
up by cancer cells, showed mixed trend of cytotoxicity in cancer
cells. Both BetA and BetA-FITC exhibited similar levels of
toxicity in selected cancer cells and no toxicities in noncancer
cells. This indicates that final fates (i.e., killing) of cells treated
with BetA or BetA-FITC are similar even though the cellular
uptake of these molecules could only be observed in cells
treated with BetA-FITC. Clearly, even though BetA-FITC and
Is-BetA exhibited more or less similar levels of entry in cancer
cells, Is-BetA could induce selectively more toxicity in cancer
cells than BetA-FITC (or BetA).
However, Is-amine induced no cytotoxicity in either cancer
or noncancer cells, except with a glaring example of cytotoxicity
in A549 cancer cells. However, as expected from the selective
uptake of Is-BetA in cancer cells, Is-BetA clearly showed
selective and consistent cytotoxicity in only cancer cells. There
were no visible Is-BetA-mediated cytotoxicities in noncancer
cells. Clearly, the conjugate (Is-BetA) is significantly more
efficient in selectively killing cancer cells when compared to the
individual partners, i.e., Is-amine and BetA. The data in Figure 1
and Table 1 clearly indicates that the fluorescent uptake of Is-
BetA was directly proportional to the killing of cells. Since
noncancer cells did not take up Is-BetA, we did not witness any
killing effect. Conversely, since cancer cells efficiently took up
Is-BetA, it induced effective killing of cancer cells. Dox is known
to have nonspecific toxicity in proliferating noncancer cells. We
obtained nonspecific uptake of Dox in all tested cells. This
might have a role in maintaining Dox-mediated indiscriminate
toxicity in both cancer and noncancer cells.
Betulinic acid has dual role in different cells. It is a known
scavenger of ROS in normal cells but induces limited ROS
generation in cancer cells. As Is-BetA exhibited efficient cancer
cell killing, it is of interest to know the status of ROS in Is-
BetA-treated cells. In fact, only Is-BetA, among its precursors
and conjugate partners, triggered excessive ROS production in
B16F10 cells (Figures 2 and S6). The ROS-generating effect of
Is-BetA in cancer cells was hence significantly and strikingly
more than that of even the ROS generating parent molecule,
Figure 2. Is-BetA selectively generates reactive oxygen species (ROS)
in cancer cells. Noncancer (NIH 3T3) and cancer cells (B16F10) were
treated with Is-PMB, Is-amine, BetA, Is-BetA, and Dox, respectively, or
kept untreated (UT). ROS as generated in respective cells were
microscopically visualized in 20× magnification.
BetA. Notably, neither Is-BetA nor its precursors Is-amine and
Is-PMB, and its conjugate partner, BetA, could generate ROS in
noncancer cells. However, ROS production was nil in Is-BetA-
treated noncancer cells (Figure S7). This could be simply
because of its least uptake in these cells. Evidently and as
expected, Dox induced the production of ROS in both cancer
and noncancer cells.
Further, using flow cytometry technique we determined the
apoptosis-inducing capabilities of tested molecules in cancer
cells. Following the treatments of respective molecules, B16F10
melanoma cells were assayed for apoptosis by doubly staining
with annexin V/propidium iodide (PI). Annexin V and PI
doubly stained cells show the characteristic of postapoptotic
cells, which contain cellular population with apoptotic and
necrotic properties simultaneously. As depicted in individual
subfigures of Figure 3, this postapoptotic population appears in
the right upper corner (Q2), whereas cells undergoing only
necrosis appear in the left upper corner (Q1), cells with
apoptotic-only feature appear in right lower corner (Q4), and
healthy cells with no apoptotic or necrotic feature appear in left
C
DOI: 10.1021/acsmedchemlett.5b00095
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ACS Medicinal Chemistry Letters
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: adhikarisusanta@yahoo.com. Phone: +91-33-2350-
8386. Fax: +91-40-2351-9755.
■
*E-mail: rkbanerjee@yahoo.com. Phone: +91-40-2719-1476.
Fax: +91-40-2719-3370.
Author Contributions
∥These authors contributed equally to this work. The
manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Funding
A.P., M.Y., and A.K.B. acknowledge fellowship supports from
CSIR, New Delhi. A.G. is thankful to UGC for a fellowship.
S.C. acknowledges DST, and A.G. and R.K. acknowledge CSIR
Network projects for respective project fellowships. This
project is supported by CSIR Network projects CSC0302,
BSC0123, Govt. of India [to R.B.] and DST project (No. SR/
S1/OC-101/2012] [to S.A.].
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge Prof. Amitabha Sarkar (IACS, Kolkata) for his
generous suggestions and support. We also thank Biswajit
Chakraborty (IICB, Kolkata) for his assistance in the isolation
of betulinic acid (in bulk quantity) from methanol extract of the
Dillenia Indica fruit commonly known as “Chalta” or Elephant
Apple.
Figure 3. Is-BetA induces apoptosis in cancer cells. Flow cytometric
analyses of B16F10 cancer cells either kept untreated (UT) or treated
with Is-PMB, Is-amine, BetA, Is-BetA, and Dox. Among all treatments,
Is-BetA-treated cells represent maximum postapoptotic features (Q2:
54.6%).
ABBREVIATIONS
BetA, betulinic acid; ROS, reactive oxygen species; Dox,
doxorubicin; FITC, fluorescein isothiocyanate
■
lower corner (Q3). It is clear that, among Is-BetA-treated cells,
maximum cancer cells exhibited features of postapoptotic
conditions. This population is significantly more than the
corresponding population from cells treated with other
individual components, Is-amine and BetA. This data is in
consonant with the significant ROS generating ability and
selectively high cytotoxicity of Is-BetA in cancer cells.
Evidently, Is-BetA-mediated excellent ROS generation in
cancer cells induced significant apoptosis in them. This could
be the primary reason for selective and efficient killing of
various cancer cells. Clearly, those cells (i.e., cancer cells),
which were fluoresced due to efficient uptake of Is-BetA,
generated huge excess of ROS and eventually were killed via
apoptosis. Hence, we accomplished the development of a single
molecule, which can effectively serve to first detect and finally
kill cancer cells, both selectively and efficiently. The drug
sensitizing property and in vivo antitumor potential of this 2-in-
1 therapeutic molecule is currently under progress.
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■
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* Supporting Information
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