Benzisothiazolones arrest the cell cycle at the G2/M phase and induce apoptosis in HeLa cells

Article abstract of DOI:10.1039/c3md00034f

Anticancer activities of a series of benzisothiazolones having alkyl, aryl and aralkyl substituents on the nitrogen atom and the mechanistic basis of cytotoxicity are presented. Cellular responses like DNA laddering, disruption of mitochondrial membrane potential and caspase-3 activation on incubation of HeLa cells with representative compounds from this group suggested the induction of apoptosis through an intrinsic pathway. Their ability to arrest the cell cycle at the G₂/M phase was confirmed by flow cytometric analysis.

Full text of DOI:10.1039/c3md00034f

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Benzisothiazolones arrest the cell cycle at the G2/M
phase and induce apoptosis in HeLa cells†
Cite this: Med. Chem. Commun., 2013,
4, 749
Pushparathinam Gopinath,‡a Krishnan Ramalingam,‡b
a
b
*
*
Kannoth Manheri Muraleedharan and Devarajan Karunagaran
Anticancer activities of a series of benzisothiazolones having alkyl, aryl and aralkyl substituents on the
nitrogen atom and the mechanistic basis of cytotoxicity are presented. Cellular responses like DNA
laddering, disruption of mitochondrial membrane potential and caspase-3 activation on incubation of
HeLa cells with representative compounds from this group suggested the induction of apoptosis
through an intrinsic pathway. Their ability to arrest the cell cycle at the G2/M phase was confirmed by
flow cytometric analysis.
Received 29th January 2013
Accepted 19th February 2013
DOI: 10.1039/c3md00034f
www.rsc.org/medchemcomm
Synthesis
Introduction
The synthesis of benzisothiazolones (BITs) 4.1 to 4.12
commenced from the commercially available dithiodibenzoic
acid (1).2 In a typical procedure, 1 was converted to dithiodi-
benzoyl chloride using thionyl chloride and then reacted with
bromine in dichloromethane to get bromosulfenylbenzoyl
chloride (2). This on treatment with aniline (3.1) in the presence
of triethylamine afforded 4.1 in 39% yield (Scheme 1). Inclusion
of other aniline derivatives with electron donating or with-
drawing substituents (3.2–3.6) in the scheme gave compounds
4.2–4.6 in 10–66% yields. Reaction of 2 with cyclohexylamine
(3.7), isobutylamine (3.8) and benzylamine (3.9) resulted in the
formation of compounds 4.7–4.9 respectively.
Compounds 4.1–4.9 were then subjected to cytotoxicity eval-
uation on HeLa (human cervical adenocarcinoma), SiHa (human
cervical squamous cell carcinoma) and SW480 (human colon
adenocarcinoma) cells using the MTT assay.25 Among these, the
cyclohexylamine and benzylamine derived benzisothiazolones,
Heterocyclic compounds containing sulfur and nitrogen atoms
are known to exhibit interesting biological properties.1 Among
these, isothiazolones and benzisothiazolones (BIT) constitute
an important class due to the presence of electrophilic sulfur as
part of the ring.2,3 The capability of these compounds to form
disulde bonds with sulfur nucleophiles or to chelate biologi-
cally relevant metals such as zinc4,5 has been correlated with
their activities against human leukocyte elastase,6–9 RNA poly-
merase, proteases,10,11 HIV-NcP-7,12 telomerase,13,14 blood
platelet aggregation,15 microbes,16–18 and proliferation.19,20
Recently, Groutas and co-workers have evaluated the activities
of benzisothiazolone derivatives against fungal strains,21 and
viruses causing Dengue and West Nile fever.22 Experiments
involving representative compounds later revealed that they
probably act by targeting the respiratory pathway and induce
the formation of reactive oxygen species.23 During our efforts to
develop new protease inhibitors based on benzisothiazolones,
we came across their anti-proliferative effect on various cancer
cell lines. As many anticancer agents are known to cause
apoptosis,24 we examined whether the most cytotoxic benziso-
thiazolones from the present study are also capable of inducing
similar effects on the susceptible HeLa cells. Results from these
cytotoxicity evaluation and cell-response studies are discussed
below.
aIndian Institute of Technology Madras, Chemistry, Chennai, India. E-mail: mkm@
iitm.ac.in
bIndian Institute of Technology Madras, Biotechnology, Chennai, India. E-mail:
karuna@iitm.ac.in
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3md00034f
Scheme 1 Synthesis of benzisothiazolone derivatives from dithiodibenzoic acid (1).
Med. Chem. Commun., 2013, 4, 749–752 | 749
‡ These authors contributed equally to this work.
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Table 1 IC50 (in mM) values of BIT derivatives in human cancer cellsa
Compounds
HeLa
SiHa
SW480
4.7
4.8
4.9
4.10
4.11
4.12
24.8
31.8
20.6
43.7
30.9
37.3
48.3
38.1
25.1
32.3
35.9
29.3
48.8
47.8
23.8
43.1
41.8
46.3
a
Cells were treated with various concentrations of BIT for 72 h using
DMSO as control and the viability was determined by the MTT assay.
Fig. 2 Gel picture showing DNA fragmentation induced by benzisothiazolones
4.7 and 4.9 in HeLa cells.
morphological and molecular changes such as cell shrinkage,
externalization of phosphatidyl serine and DNA fragmenta-
tion.26 Based on the sequence of events involved, apoptosis can
be extrinsic or intrinsic.27 The former usually is initiated by the
interaction of extracellular factors such as TNF (Tumor Necrosis
Factor) with the death receptors and the subsequent propaga-
tion of signals via activation of caspase-8. The intrinsic pathway
on the other hand occurs aer the internalization of cytotoxic
agents through the cell membrane. Their interaction with
cellular targets then activates specic signals that prepare the
cells for programmed cell death. Since the apoptotic response of
cancer cells aer benzisothiazolone treatment has never been
studied systematically, we carried out a series of experiments
and the results are outlined below.
The cleavage of nuclear DNA into oligonucleosomal frag-
ments usually occurs in response to apoptotic stimuli. Since
these fragments appear like a ladder in electrophoretic gels, it is
termed as DNA laddering and is considered a hallmark of
apoptosis.28 DNA fragmentation assay results aer incubating
compounds 4.7 and 4.9 with HeLa cells for 72 h are presented in
Fig. 2. DNA was stained with ethidium bromide aer electro-
phoresis on a 2% agarose and then visualized under UV light. In
Fig. 2, lane 1 represents the DNA marker whereas lanes 2 and 3
show the stained DNA in cells treated with DMSO (control) and
taxol, respectively. The results aer treating HeLa cells with
compounds 4.9 (50 mM) and 4.7 (50 mM) shown respectively in
lanes 4 and 5 suggest that both 4.9 and 4.7 induce DNA
fragmentation.
Fig. 1 Bar diagram showing the effect of NAC and DTT on the anticancer activity
of 4.9.
4.7 and 4.9, showed IC50 values of 24.8 and 20.6 mM against HeLa
cells (Table 1). They respectively showed IC50 values of 48.3 mM
and 25.1 mM against SiHa cells, and 48.8 mM and 23.8 mM against
SW480 cell lines. The corresponding values for 4.8 were slightly
higher and ranged between 31.8 and 47.8 mM. The aniline
derivatives 4.1–4.6 although soluble in DMSO started precipi-
tating (visible only under a microscope) in the cell culture
medium and hence were not taken forward for the estimation of
IC50 values. Based on the structure of 4.9, a second line of
derivatives were then prepared and evaluated against all the three
cell lines (4.10–4.12). Since these compounds did not show
signicant improvement in their potencies in comparison with
4.7 and 4.9 (Table 1) the latter two were selected for detailed
mechanistic investigations in HeLa cells.
As mentioned, benzisothiazolones possess an electrophilic
sulfur having a high tendency to form adducts with compounds
containing thiol functionalities. In order to see whether such a
reaction is responsible for the observed cytotoxicity, we co-
administered N-acetyl cysteine (NAC) along with the compound
4.9 during the MTT assay. As shown in Fig. 1, this completely
abolished the cytotoxicity (compare bars 3 and 5 in Fig. 1)
suggesting the possibility of such covalent adduct formation
with important cellular targets. A comparable result was
obtained on using dithiothreitol (DTT) in place of NAC
(compare bars 3 and 8 in Fig. 1).11
Apoptosis induction is also associated with the disruption of
mitochondrial membrane potential and release of Cytochrome
c into the cytosol. The state of the membrane can be followed
using cationic JC1 dye having a tendency to accumulate in the
interior of mitochondria.29 JC1 in the monomeric form emits in
the green region, whereas its aggregates will be orange/red.30
This dye aggregates in mitochondria with normal membrane
potential but remains monomeric when the potential is low. As
shown in Fig. 3A, untreated HeLa cells appeared orange but
treatment with taxol induced noticeable disruption of the
membrane potential as indicated by emission in the green
Mechanistic insight into the cytotoxic effect by 4.7 and 4.9
Small-molecule anticancer agents usually exert a cytotoxic effect region (Fig. 3B). The response from compounds 4.9 and 4.7 was
by inducing apoptosis, process characterized by both similar to that of taxol, which suggested signicant disruption
a
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Fig. 5 HeLa cells treated with the 4.9 at the indicated concentration for 48 h
were lysed. The lysates were resolved on a 10% SDS-PAGE, transferred on to a
nitrocellulose membrane and probed for cleaved caspase-3. The same blot was
then re-probed for actin.
these experiments are summarized in Fig. 4A–C which show the
accumulation of cells in the G2/M phase, depletion of cells in
the G0/G1 phase and an increase in apoptotic fraction in their
presence. Exposure of HeLa cells to compounds 4.9 and 4.7 at a
50 mM concentration for 48 h resulted in 18% and 23.4% of
apoptotic cells respectively, whereas only 7.3% of apoptosis
could be seen in untreated cells (Fig. 4B). Similarly, there were
18.6% and 26.6% of the G2/M phase cells in these cases
compared to 14.3% for untreated cells (Fig. 4C).
Apoptosis signals will nally lead to the processing of pro-
caspase-3 to caspase-3,33 which then assists the degradation of
cellular proteins. We have looked for the presence of caspase-3
aer treating HeLa cells with 4.9. The western blot showing the
presence of cleaved caspase-3 aer incubating HeLa cells with
taxol (12 nM) and 4.9 (50 mM) is shown in Fig. 5. The presence of
this enzyme in cells treated with 4.9 clearly shows the process-
ing of pro-caspase-3 and induction of apoptosis.
Fig. 3 (A) JC-1 localized in the mitochondria of HeLa cells, (B) HeLa cells after the
treatment of 12 nM taxol, (C and D) HeLa cells treated with 4.9 (50 mM), and 4.7
(50 mM), respectively.
of the potential at the concentration tested (Fig. 3C and D). To
understand the effect of 4.9 and 4.7 on the cell cycle,31 they were
incubated with HeLa cells for 24 h, xed, stained using propi-
dium iodide and analyzed by ow-cytometry.32 The results of
As mentioned, apoptosis induction is characteristic of a
number of known anticancer agents.24 While the effect of taxol
is obvious from control experiments presented here,
compounds such as mimosine34 (which interfere with DNA
replication and cause oxidative stress) are also known to trigger
all the cellular responses typical of apoptosis discussed here.
Apart from benzisothiazolones, our laboratory has investigated
the anticancer activities of a number of redox-active 1,2-dihy-
droquinolines. Some compounds from this group, which
showed low micromolar IC50 values, have also shown compa-
rable effects.35
Conclusion
In conclusion, we have synthesized and evaluated the anti-
cancer activities of a number of benzisothiazolones. The
importance of electrophilic sulfur for their anticancer activity
became evident when they were co-administered with NAC or
DTT during the MTT assay. Using representative derivatives, we
have shown that they induce apoptosis characterized by cellular
responses like DNA fragmentation, disruption of mitochondrial
membrane potential and activation of caspase-3. The subse-
quent ow cytometric analysis involving the same compounds
(4.7 and 4.9) clearly showed that they are capable of arresting
the cell cycle at the G2/M phase. The results presented here raise
Fig. 4 (A) X-axis indicates the DNA content and Y-axis indicates the cell count;
each graph shows the distribution of the G0/G1 phase, S phase, G2/M phase and
apoptotic levels. Data represent the mean Æ SEM of three independent experi-
ments. (B) % of apoptotic cells and (C) % of the G2/M phase cells.
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the hope that this class of compounds can be developed into 14 F. Lavelle, J. F. Riou, A. Laoui and P. Mailliet, Crit. Rev. Oncol.
new anticancer leads by proper structure optimization.
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Acknowledgements
16 F. Zani, P. Vicini and M. Incerti, Eur. J. Med. Chem., 2004, 39,
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17 F. Zani, M. Incerti, R. Ferretti and P. Vicini, Eur. J. Med.
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Financial support of this work by CSIR, INDIA (Grants 01(2121)/
07/EMR-II), DST, INDIA (Grants SR/S1/OC-13/2007) and by DBT,
INDIA (Grants BT/01/COE/07/04) is gratefully acknowledged. PG
thanks IIT-Madras for Research Fellowship.
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