Mitochondrial Impairment by Cyanine-Based Small Molecules Induces Apoptosis in Cancer Cells

Article abstract of DOI:10.1021/acsmedchemlett.9b00304

Mitochondrion, the powerhouse of the cells, has emerged as one of the unorthodox targets in anticancer therapy due to its involvement in several cellular functions. However, the development of small molecules for selective mitochondrial damage in cancer c

Full text of DOI:10.1021/acsmedchemlett.9b00304

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Mitochondrial Impairment by Cyanine-Based Small Molecules
Induces Apoptosis in Cancer Cells
Sohan Patil,† Deepshikha Ghosh,§ Mithun Radhakrishna,∥ and Sudipta Basu*,‡
†Department of Chemistry, Indian Institute of Science Education and Research (IISER)-Pune, Dr. Homi Bhabha Road, Pashan,
Pune, Maharashtra 411008, India
§
‡Discipline of Chemistry, Discipline of Biological Engineering, and∥Discipline of Chemical Engineering of Indian Institute of
Technology (IIT)-Gandhinagar, Palaj, Gandhinagar, Gujarat 382355, India
S
* Supporting Information
ABSTRACT: Mitochondrion, the powerhouse of the cells, has emerged as one of the unorthodox targets in anticancer therapy
due to its involvement in several cellular functions. However, the development of small molecules for selective mitochondrial
damage in cancer cells remained limited and less explored. To address this, in our work, we have synthesized a natural product
inspired cyanine-based 3-methoxy pyrrole small molecule library by a concise strategy. This strategy involves Vilsmeier and
Pd(0) catalyzed Suzuki cross-coupling reactions as key steps. The screening of the library members in HeLa cervical cancer cells
revealed two new molecules that localized into subcellular mitochondria and damaged them. These small molecules perturbed
antiapoptotic (Bcl-2/Bcl-xl) and pro-apoptotic (Bax) proteins to produce reactive oxygen species (ROS). Molecular docking
studies showed that both molecules bind more tightly with the BH3 domain of Bcl-2 proteins compared to obatoclax (a pan-
Bcl-2 inhibitor). These novel small molecules arrested the cell cycle in the G0/G1 phase, cleaved caspase-3/9, and finally
prompted late apoptosis. This small molecule-mediated mitochondrial damage induced remarkably high cervical cancer cell
death. These unique small molecules can be further explored as chemical biology tools and next-generation organelle-targeted
anticancer therapy.
KEYWORDS: Mitochondria, Suzuki cross-coupling, cell cycle arrest, apoptosis, cancer
perturb mitochondrial translational machinery, ion channels,
itochondrion (the powerhouse of the cells) is one of the
Mmost central organelles which controls ATP (energy fission-fusion, and heat-shock proteins.19−22 Hence, there is an
urgent need to develop novel and easily synthesizable small
molecules to impair mitochondria in cancer cells for next-
generation anticancer therapy.23
To address this, we report a short and concise synthesis of
cyanine-based 3-methoxy pyrrole derivatives. 3-Methoxy
pyrrole is a privileged structure present in the Prodigiosin
family of natural products with a broad range of anticancer
activities.24−29 We introduce a cyanine moiety due to its
presence in MitoTracker Deep Red (mitochondria localizing
dye) (Scheme 1a). Screening of these small library members in
currency) synthesis, retro-/anterograde signaling, biosynthesis,
stress response, and storage of genomic materials.1−5 Different
stages of cancer development and progression involve
alteration in mitochondrial functions.6−9 Hence, targeting
mitochondrial proteins and genomic materials has emerged as
an unconventional strategy in cancer therapy.10−13 Clinically
approved small molecule anticancer drugs have been chemi-
cally tagged with cationic moieties (triphenylphosphonium ion
and peptides) to detour them into mitochondria. This strategy
improved drug efficacy, overcame drug resistance, and reduced
off-target adverse effects.14−18 Nevertheless, the development
of novel small molecules to target mitochondrial functions in
cancer cells is still in its infancy. In this direction, a few small
molecules have been identified and synthesized to directly
Received: July 8, 2019
Accepted: December 10, 2019
Published: December 10, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.9b00304
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(7a−p) was synthesized (Table 1, Figure S3). The chemical
structures of all the intermediates and the final library members
Scheme 1. (a) Chemical Structures of 3-Methoxy-Pyrrole
Based Biologically Active Natural Products, MitoTracker
Deep Red, and Focused Library. (b) Synthetic Scheme of
Cyanine-Based 3-Methoxy-Pyrrole Library. (c) Schematic
Representation of Compounds 7n and 7p Impairing
Mitochondria in Cancer Cells toward Apoptosis. (d)
ORTEP Diagram of Compound 7d with 50% Thermal
Ellipsoids
Table 1. Compound Library (7a−p)
1
were confirmed by H, 13C NMR, and HR-MS spectroscopy
(Figure S4−S102). Furthermore, we also confirmed the
structure of one of the library members (7d) by X-ray
crystallography (Scheme 1d, Table S1). Interestingly, all the
library members were found to be red fluorescent in nature
with a range of emission from λmax = 556−593 nm (Figure
S103, Table S2). This result was suitable for subcellular
visualization.
HeLa cervical cancer cells identified two highly potent new
small molecules which localized into mitochondria and
impaired them. These novel lead molecules inhibited
antiapoptotic Bcl-2/Bcl-xl, activated pro-apoptotic Bax, and
generated reactive oxygen species (ROS). These novel small
molecule-mediated mitochondrial damages triggered cell cycle
arrest in the G0/G1 phase and induced late apoptosis. This
mitochondrial damage resulted in remarkable HeLa cervical
cancer cell death at submicromolar concentration compared to
clinically approved drugs (Scheme 1c).
The synthesis of cyanine-based 3-methoxy pyrrole is
illustrated in Scheme 1b. We first reacted 4-methoxy-3-
pyrroline-2-one (1) with N,N-diethyl formamide (2) in the
presence of phosphorus oxybromide to obtain the 5-bromo-3-
methoxy pyrrolidine derivative (3) (yield = 60%) through a
modified Vilsmeier reaction. Compound 3 was then exposed to
C−C bond formation by Suzuki cross-coupling reaction. We
reacted compound 3 with aromatic boronic acids (4a−p)
(Figure S1), in the presence of tetrakis(triphenylphosphine)
palladium(0) as a catalyst to afford aromatic substituted 3-
methoxy-pyrrole aldehydes (5a−p) in 70−90% yield (Figure
S2). Finally, the aldehydes were condensed with 1,2,3,3-
tetramethyl-3-H-indolium iodide (6) in the presence of acetic
anhydride as a dehydrating agent to achieve focused library
members. A library of 16 cyanine-based 3-methoxy-pyrroles
We tested the library members for their cancer cell killing
ability. We incubated HeLa cervical cancer cells with
compounds 7a−7p in a concentration-dependent manner for
24 h. Excitingly, the library members showed remarkable dose-
dependent HeLa cell killing ability with IC50 values ranging
from 4.47 to 0.92 μM (Figure 1, Table S3). We chose
compounds 7n and 7p for further studies due to their best cell
killing ability with IC50 = 0.97 and 0.92 μM, respectively. We
also compared the efficacy of compounds 7n and 7p with
clinically approved anticancer drugs (obatoclax, cisplatin,
SN38, doxorubicin, and paclitaxel) in a dose-dependent
manner for 24 h by MTT assay. To our delight, we found
that cisplatin and SN38 showed negligible cell death even at 10
μM concentrations. However, obatoclax and paclitaxel showed
moderate activity with cell viability of 70.35 5.0% and 68.74
3.2%, respectively (Figure S104). Only doxorubicin
demonstrated comparable cancer cell killing with IC50 = 2.0
μM, that is as per our previous study.30,31 We further evaluated
the effect of 7n and 7p in noncancerous HEK293 human
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Figure 2. (a) Confocal laser scanning microscopy images of HeLa
cells incubated with compounds 7n and 7p for 20 min. The
mitochondria were stained with green fluorescent MitoTracker Green
dye. The colocalization of compounds 7n and 7p was seen by merged
yellow regions. (b) Intensity scatter plots of compounds 7n and 7p
showing colocalization into mitochondria from confocal microscopy.
Figure 1. Dose-dependent cell viability assay of all the library
members in HeLa cells at 24 h postincubation.
embryonic kidney cells for 24 h postincubation. We found that
7n and 7p showed much higher IC50 values of 2.56 and 2.61
μM with 24 and 25% of cell viability (at 5 μM), respectively,
compared to HeLa cells (Figure S105).
and 7p induced mitochondrial damage by opening MPTPs in
HeLa cells.
We hypothesized that these novel cyanine-based 3-methoxy-
pyrroles would induce mitochondrial impairment. To validate
our hypothesis, we first observed the subcellular localization of
compounds 7n and 7p into mitochondria by confocal laser
scanning microscopy. HeLa cells were incubated with
compounds 7n and 7p at 150 nM concentration for 20 min,
followed by staining mitochondria by green fluorescent
MitoTracker Green dye. The live HeLa cell imaging under
confocal microscopy revealed that red fluorescent compounds
7n and 7p localized into green fluorescently tagged
mitochondria to yield merged yellow signals (Figure 2a, Figure
S106a). Intensity scatter plots of both the compounds also
confirmed remarkable colocalization (Figure 2b). Further, z-
stack maximum projection images obtained from confocal
imaging (Figure S106b) also validated that compounds 7n and
7p homed into mitochondria of HeLa cells within 20 min.
When inside mitochondria, the lead compounds 7n and 7p
should open up mitochondrial permeability transition pores
(MPTPs), leading to the reduction in mitochondrial
membrane potential (ΔΨm) followed by rupture of the
outer membrane.32 We evaluated the opening of MPTPs by
compounds 7n and 7p by Calcein AM assay.33 To estimate
MPTP formation, we treated HeLa cells with compounds 7n
and 7p for 24 h followed by incubation with Calcein AM and
CoCl2. The live HeLa cells were visualized by confocal
microscopy. The confocal images (Figure 3a, S107a) of
nontreated control cells hardly showed any green fluorescent
signal. On the other hand, compound 7n and 7p treated cells
demonstrated a remarkable increase in green fluorescence
intensity (3.6-fold and 3.9-fold, respectively, Figure S107b),
consistent with our previous observation of MPTP forma-
tion.34,23 These confocal images confirmed that compounds 7n
We further evaluated the morphological change as an
indication of mitochondrial damage by 7n and 7p.23 HeLa cells
were treated with 500 nM of 7n and 7p for 3 h. The
mitochondrial morphology was visualized by confocal imaging.
The control cells were stained with MitoTracker Green. From
the confocal imaging, we observed that 7n and 7p indeed
damaged the mitochondria into smaller fragments (Figure
S108a). In contrast, we observed elongated and filamentous
mitochondrial morphology in the control cells. Further
quantification from confocal microscopy also revealed that
7n and 7p induced the reduction in the average mitochondrial
area by 3.4-fold and 2.6-fold, respectively, compared to control
cells (Figure S108b). These confocal images confirmed that 7n
and 7p damaged mitochondria.
The mitochondrial damage through MPTP formation was
triggered by suppression of antiapoptotic Bcl-2/Bcl-xl proteins
and activation of pro-apoptotic Bax.35 Hence, we evaluated the
expression of Bcl-2, Bcl-xl, and Bax by confocal microscopy.
HeLa cells were treated with compounds 7n and 7p for 24 h
followed by incubation with Bcl-2, Bcl-xl, and Bax primary
antibodies. These primary antibodies were further recognized
by Alexa Fluor 488 tagged (green-fluorescent) secondary
antibody. The nuclei of the cells were counterstained by DAPI
(blue). Fluorescence confocal microscopy showed high green
fluorescence in control cells, confirming the presence of Bcl-2
and Bcl-xl (Figure 3b, c, Figure S109a, Figure S110a).
However, treatment of compounds 7n and 7p showed a
remarkable reduction in green fluorescence intensity into HeLa
cells. This result validated the inhibition of antiapoptotic Bcl-2
and Bcl-xl. Further quantification from confocal images
confirmed that compound 7n reduced the green fluorescence
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the binding affinities of 7n and 7p in the BH3 domain of Bcl-2
were found to be −12 and −12.4 kcal/mol, respectively. These
binding affinities were higher than the binding affinity of
obatoclax (−6.5 kcal/mol) (Table S4). These docking studies
confirmed that 7n and 7p binding with the BH3 domain of
Bcl-2 was stronger than obatoclax leading to improved
inhibition and cell killing.
This small molecule-mediated mitochondrial damage would
lead to the generation of reactive oxygen species (ROS).28 We
evaluated ROS generation by 2′,7′-dichlorodihydrofluorescein
(H2DCFDA) assay.39 HeLa cells were treated with compounds
7n and 7p, followed by incubation with H2DCFDA. We then
visualized the cells under a fluorescence microscope. The
confocal images (Figure S113a) showed that control cells
hardly generated any green fluorescent signals indicating
negligible ROS generation. However, compound 7n and 7p
treated cells showed a remarkable increase in green fluorescent
signal intensity (16.1-fold and 22.4-fold, respectively) confirm-
ing the production of subcellular ROS (Figure S113b). This
microscopy assay established that compounds 7n and 7p
generated reactive oxygen species upon mitochondrial impair-
ment.
Mitochondrial damage through inhibition of Bcl-2/Bcl-xl
and ROS generation led to cell cycle arrest.40 We treated HeLa
cells with compounds 7n and 7p for 24 h and stained cellular
DNA by propidium iodide (PI). We assessed the cells in
different phases of the cell cycle by flow cytometry. The flow
cytometry data revealed that 4.7%, 67.9%, 6.4%, and 16.8% of
the cells were in the apoptotic, G0/G1, S, and G2-M phase
after treatment with compound 7n, respectively. In contrast,
only 1.2%, 47.4%, 13.8%, and 33.9% of the cells were found in
the apoptotic, G0/G1, S, and G2-M phase, respectively, for
control cells (Figure 4a). Similarly, after treatment with
compound 7p, 3.5%, 63.9%, 13.5%, and 15.0% of the cells
were found in the apoptotic, G0/G1, S and G2-M phase,
respectively. This flow cytometry analysis exhibited that both
compounds 7n and 7p induced cell cycle arrest in the G0/G1
phase after mitochondrial damage.
Mitochondrial damage and cell cycle arrest would trigger
programmed cell death or apoptosis.40,41 We quantified the
induction of apoptosis by flow cytometry after treatment of
HeLa cells with compounds 7n and 7p for 24 h. We further
stained phosphatidylserine (at the outer membrane of
apoptotic cells) and nuclear DNA (in necrotic cells) by
Allophycocyanine (APC)-tagged Annexin V and 7-amino-
actinomycin D (7-AAD), respectively. The cells were then
sorted by flow cytometry, which demonstrated that both
compounds 7n and 7p remarkably induced 99.5% and 98.7%
cells into the late apoptotic stage, respectively (Figure 4b). The
induction of late apoptosis was further validated by Western
blot analysis. We evaluated the cleavage of initiator caspase-9
and executioner caspase-3 as apoptosis markers.42,43 We
incubated HeLa cells with compounds 7n and 7p for 24 h,
and the whole-cell proteins were subjected to gel electro-
phoresis. The Western blot images explicitly showed that the
expression of caspase-3 was reduced significantly to 3.5-fold
and 3.7-fold by compounds 7n and 7p, respectively, compared
to control cells (Figure 4c, Figure S114a). Similarly,
compounds 7n and 7p also reduced the expression of
caspase-9 to 1.9- and 2-fold, respectively (Figure 4c, Figure
S114b). These flow cytometry and gel electrophoresis analyses
confirmed that compounds 7n and 7p activated late apoptosis
through cleavage of the caspase-3/9-based intrinsic pathway.
Figure 3. Confocal microscopy images of HeLa cells treated with
compounds 7n and 7p for 24 h followed by (a) staining with Calcein
AM to observe mitochondrial permeability transition pore opening
(MPTPs). (b−d) Staining with Bcl-2, Bcl-xl, and Bax primary
antibodies and green fluorescent Alexa Fluor 488-secondary antibody,
respectively. Nuclei were stained with blue fluorescent DAPI dye.
Scale bar = 10 μm. (e) Western blot analysis of Bcl-2, Bcl-xl, and Bax
in HeLa cells after treatment with compounds 7n and 7p for 24 h.
intensity for Bcl-2 and Bcl-xl by 8.5-fold and 10.1-fold,
respectively (Figure S109b, Figure S110b). Compound 7p also
reduced the green fluorescence intensity for Bcl-2 and Bcl-xl by
5.7-fold and 8.4-fold, respectively, compared to control cells
(Figure S109b, Figure S110b). On the other hand, confocal
images (Figure 3d, Figure S111a) and quantification (Figure
S111b) showed that compounds 7n and 7p increased the
expression of pro-apoptotic Bax proteins by 2.1-fold and 2.4-
fold, respectively, compared to control cells.
We validated the inhibition of Bcl-2/Bcl-xl and activation of
Bax by gel electrophoresis. HeLa cells were treated with
compounds 7n and 7p for 24 h, and the cellular proteins were
subjected to Western blot analysis. The Western blot images
and quantification showed that compound 7n reduced the
expression of Bcl-2 and Bcl-xl by 6.4-fold and 4.0-fold,
respectively (Figure 3e, Figure S112a,b). Compound 7p
treatment reduced the expressions of Bcl-2 and Bcl-xl by 9.3-
fold and 5.6-fold, respectively (Figure 3e, Figure S112a,b).
However, the expression of Bax was remarkably increased by
compounds 7n and 7p by 3.6-fold and 4.1-fold, respectively
(Figure 3e, Figure S112c). These confocal microscopy and
Western blot studies confirmed that compounds 7n and 7p
inhibited antiapoptotic Bcl-2/Bcl-xl and activated pro-
apoptotic Bax proteins, which led to mitochondrial damage.
Compounds 7n and 7p are structurally similar to obatoclax,
which binds with the BH3 domain of Bcl-2 proteins.36 Hence,
we carried out molecular docking studies using Auto Dock
Vina in conjunction with MGL, ADT, and PMV software to
quantify the binding affinity.37,38 From these docking studies,
D
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ACS Medicinal Chemistry Letters
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Western blots, crystal data, photophysical data, and IC50
(PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: sudipta.basu@iitgn.ac.in.
■
ORCID
Sohan Patil: 0000-0003-0940-9948
Mithun Radhakrishna: 0000-0001-7127-4744
Sudipta Basu: 0000-0002-0433-8899
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project was financially supported by the Department of
Biotechnology (BT/RLF/Re-entry/13/2011 and BT/
PR14724/NNT/28/831/2015) and Department of Science
and Technology, Govt. of India [SB/NM/NB-1083/2017
(G)]. S. Patil sincerely thanks CSIR-UGC for a fellowship. We
sincerely thank Ravindra Raut of IISER-Pune for helping in X-
ray crystallography analysis. We also thank Monal Desai and
Akanksha Tripathi of the Writing Studio, IIT-Gandhinagar for
helping in manuscript preparation.
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00304.
■
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DOI: 10.1021/acsmedchemlett.9b00304
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX