Fluorinated thiazolidinols cause cell death in A549 lung cancer cells: Via PI3K/AKT/mTOR and MAPK/ERK signalling pathways

Article abstract of DOI:10.1039/c5md00603a

A series of 2-imino-4-(trifluoromethyl)thiazolidin-4-ol derivatives were synthesized from one pot, three-component reactions of primary amine, aryl isothiocyanate and 3-bromo-trifluoromethyl acetone via in situ generation of both symmetrical and unsymmetr

Full text of DOI:10.1039/c5md00603a

View Article Online
View Journal
MedChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: R. M. Kumbhare,
T. L. Dadmal, D. Kumar, J. ramana, A. Kota, D. C. chowdhury, K. appalanaidu, Y. K. Rao, S. Hyder, T. A. Devi
and M. P. Bhadra, Med. Chem. Commun., 2016, DOI: 10.1039/C5MD00603A.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/medchemcomm

MedChemComm
Page 1 of 8Journal Name
Dynamic Article Links ►
View Article Online
DOI: 10.1039/C5MD00603A
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
ARTICLE TYPE
Fluorinated thiazolidinols cause cell death in A549 lung cancer cells
via PI3K/AKT/mTOR and the MAPK/ERK signalling pathways†
Ravindra M. Kumbhare,*a Tulshiram L. Dadmal,a,d Dinesh Kumar,b M. Janaki Ramaiah,c Anudeep
Kota, Debabrata Chowdhury,b K. Appalanaidu,a Y. Khageswara Rao,a Sayyad Hyder,a T. Anjana
Devi,*b and Manika Pal Bhadra*b
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
A series of 2ꢀiminoꢀ4ꢀ(trifluoromethyl)thiazolidinꢀ4ꢀol derivatives were synthesized from one pot, three component reactions of primary
amine, aryl isothiocyanate and 3ꢀbromoꢀtrifluoromethyl acetone via in situ generation of both symmetrical and unsymmetrical thioureas.
10 All the synthesized derivatives were screened for their in vitro antiꢀcancer activity against human cancer cell lines. Compounds 8l, 8r, 8s,
8t and 8u exhibited potent anticancer activity in lung cancer cells (A549) than the other three cell lines. Data obtained from analysis of
cell cycle showed that treatment of lung cancer cells with these compounds resulted in G0/G1cell cycle arrest. Studies to understand the
molecular mechanism of action of these compounds suggest that the compounds inhibit PI3K, pAkt and mTOR protein expression with
concomitant up regulation of tumor suppressor PTEN. These compounds contributed to LCꢀ3 mediated cytoplasmic vacuolation leading
15 to cell death in lung cancer cells. Overall, these compounds modulate the cell death processes by acting via inhibition of
PI3K/Akt/mTOR pathway and MEK/ERK pathway, key growth factor signalling pathways implicated in abnormal cell proliferation.
These molecules can therefore be further tested in in vivo models as a potential regimen for efficacy and effectivity in lung cancer.
50 survival and motility. PI3K signalling is implicated in various
diseases such as cancer, chronic inflammation, allergies and
metabolic disorders2. In addition, aberrant activation of PI3K
signalling confers resistance to conventional therapies. Thus,
PI3K is an attractive molecular target for novel antiꢀcancer
55 therapeutics against many cancers including lung cancer3,4. In
most cancer cells, elevated activity of PI3K/mTOR is observed.
In addition, RAS/RAF/ERK pathway is activated which acts in
parallel to promote cell survival. Combinations of MEK/ERK
inhibitors with inhibitors of PI3K/mTOR have proved to be more
60 effective than single agents in mouse models of KRASꢀdriven
Introduction
Lung cancer is the most common cause of cancer related
20 mortality. Despite significant research progress there remains
many obstacles in development of effective therapy against lung
cancer. Cancer chemotherapy is limited by various drug
resistance mechanisms. The cellular response towards cytotoxic
drugs is regulated by crossꢀtalk between oncogenic signalling
25 pathways and resistance mechanisms. Oncogenic signalling
pathways are typically activated by over expression of tyrosine
kinase receptors or permanant activation of downstream kinase
pathways such as the PI3K/ Akt and MEK/ MAPK pathways
which result in malignancy via inhibition of cell death process1.
30 Phosphatidylinositolꢀ3ꢀkinases (PI3Ks) are lipid kinases that
regulate diverse cellular processes including proliferation,
lung cancer5,6
.
In mammalian cells, different types of programmed cell death
have been identified7. Among them typeꢀI and typeꢀII cell deaths
are found to be major processes. Type I cell death corresponds to
65 apoptotic cell death characterized by cell shrinkage, chromatin
condensation and degradation of nucleosomal DNA of cells into
apoptotic bodies. In contrast, typeꢀII cell death or autophagic cell
death is characterized by appearance of double or multiple
membrane cytoplasmic vesicles engulfing cytoplasm or
70 cytoplasmic organelles such as mitochondria and endoplasmic
reticulum. Furthermore, these autophagic vesicles and their
contents are destroyed by lysosomes8. Other than these two
mechanisms there are mechanisms such as non apoptotic, non
autophagic cell death characterised by LCꢀ3 mediated extensive
75 cytoplasmic vacuolation.
a
Fluoroorganic Division, Indian Institute of Chemical Technology,
Tarnaka, Hyderabad, 500607, India. Fax: +91 40 27193185; Tel: +91 40
35 27191776 E-mail: kumbhare@iict.res.in
b Centre for Chemical Biology, Indian Institute of Chemical Technology,
Tarnaka, Hyderabad, 500607, India. Tel: 91 40 27193236; E-mail:
manika@iict.res.in
bCentre for Chemical Biology, Indian Institute of Chemical Technology,
40 Tarnaka, Hyderabad, 500607, India. Tel: 91 40 27191862;
E-mail: anjana@iict.res.in
cSchool of Chemical and Biotechnology, Sastra University,
Thirumalaisamdram, Tanjavur-613402, Tamil nadu.
dGovernment of Maharashtra’s, Ismail Yusuf College, Jogeshwari (East),
45 Mumbai 400060, India
† The authors declare no competing interests.
Electronic Supplementary Information (ESI) available: [Spectral data of
Compounds 8a–v, and experimental procedures for synthesis and
biological evaluations]. See DOI: 10.1039/b000000x/
It is well known that the introduction of fluorine atom or
fluorinated functional group at a specific position is of much
interest in modern era of medicinal chemistry for modulating the
physical and biological properties of the molecule9.
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1

MedChemComm
Page 2 of 8
View Article Online
DOI: 10.1039/C5MD00603A
Trifluoromethylation is one of the most significant strategies to
The 2ꢀiminoꢀtrifluoromethyl thiazolidinol derivatives (8a-v) were
improve pharmacological activities of the molecule. This is due 60 synthesized by the reaction of various amine, substituted
to its high lipophilicity, which enhances in vivo uptake as well as
phenylisothiocyanates
and
3ꢀbromoꢀ1,1,1ꢀtrifluoromethyl
transport of molecules to the active site of human body. Thus,
propanone in DMF as solvent at room temperature27. The reaction
proceeded through in situ generation of symmetrical and
unsymmetrical thioureas, as an exothermic reaction at room
5
fluorine chemistry has got lot of demand in drug discovery10,11
.
In view of the fact that heterocyclic compounds such as
thiazolidines, thiazolines and thiazolidinones are considered 65 temperature between aromatic and aliphatic amines and
privileged structural fragments in medicinal chemistry which
account for their broad pharmacological activities and possess
substituted phenylisothiocyanates, followed by reaction with 3ꢀ
bromoꢀ1,1,1ꢀtrifluoromethyl acetone, resulting in formation of
product 8a-v (scheme 1).
10 affinity for various biological targets12ꢀ14, the search for new
strategies towards the latter entities is of much significance. 2ꢀ
Iminoꢀ1,3ꢀthiazolidines and unsaturated thiazolines are known in
pharmaceutical chemistry for their antiꢀinflammatory,
antimicrobial, antihistamic, antihypertensive, anticonvulsant,
70
75
80
85
90
15 anodyne,
antiꢀAlzheimer
properties15,16
.
In
particular,
thiazolidinones are peroxisome proliferator activated receptor
(PPARꢀreceptor) agonists with antineoplastic and antitumor
properties17,18. One such example is 5ꢀbromoꢀ3ꢀ[(3ꢀsubstitutedꢀ5ꢀ
methylꢀ4ꢀthiazolidinoneꢀ2ꢀylidene hydrazono]ꢀ1Hꢀ2ꢀindolinones
20 1 (Fig. 1) which have been investigated for their primary
cytotoxic activity. It was observed that thiosemicarbazone
derivatives of indolinones show promising cytotoxicity19. Among
the 4ꢀthiazolidinone compounds, 2 and 3 (Fig. 1) possess high
antimitotic effect in vitro in submicromolar concentrations (7 and
25 10 µ M) and are characterized by low in vivo toxicity20,21
.
Micacocidin 4, a natural product based thiazolidine is a highly
cytotoxic compound with metal chelating capability, isolated
from the cultured broth of Pseudomonas sp. which also showed
the anticancer activity22,23
.
30
In continuation of our studies on development of novel
antitumor agents24ꢀ26 and keeping in mind the biological
importance of thiazolidines, thiazolines and thiazolidinones, we
planned to insert a trifluoromethyl group into the thiazolidine ring
to achieve better activity. Therefore, in the current study we have
Scheme 1 Synthesis of 2ꢀiminoꢀtrifluoromethyl thiazolidinol derivatives
35 synthesized and evaluated the biological activities of certain
novel 2ꢀiminoꢀ4ꢀ(trifluoromethyl) thiazolidinꢀ4ꢀol derivatives via
one pot, three component reaction and by a catalyst free method.
Biological evaluation indicated that the effective compounds
have the ability to induce LCꢀ3 mediated cell death in lung cancer
40 cells and act as inhibitors of PI3K/Akt/mTOR and MEK/ERK
pathway as well.
Biological evaluation
95 2-imino-trifluoromethyl thiazolidinol derivatives affect cell
viability. Cancer cells exhibit uncontrolled cell proliferation
while antiꢀcancer drugs show effect on cell viability and cause
death of the cancer cells by various mechanisms. Thus, in order
to study the plausible cytotoxic role of these molecules on A549,
100 MDAꢀMBꢀ231, MCFꢀ7 and HeLa, these cancer cell lines were
treated with the compounds for 24h and the cell viability was
determined using MTT assay. The PI3K inhibitor LY294002(Ly)
was used as the standard. We observed that the compounds 8l, 8r,
8s, 8t, 8u have shown strong cytotoxicity than others in the series
105 tested. Interestingly, these active molecules have ꢀCF3 in R1
position that might be a contributing factor for the cytotoxic
effect. Among all compounds, 8l has shown significant effect on
cell viability of A549 cells (Table 1).
45
50
110 Table 1 In vitro cytotoxicity of 2ꢀiminoꢀtrifluoromethyl thiazolidinol
55
derivatives (8a–v) against A549, MDAꢀMBꢀ231, HeLa and MCFꢀ7
Fig. 1 Biologically active thiazolidine derivatives
cancer cells.
IC50 (µM)
Results and discussion
Cmpds
A549
MDA-MB-
231
HeLa
MCF-7
Chemistry
2
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 3 of 8
MedChemComm
View Article Online
DOI: 10.1039/C5MD00603A
8a
8b
8c
8d
8e
11.05 +0.37
8.26 + 0.82
10.61+ 0.65
13.77+ 0.43
15.08+ 0.38
23.13 + 0.92 24.78 +0. 95 33.76 + 1.88
21.57 + 1.77 24.82 + 1.53 29.41 + 1.42
20.58 + 1.24 27.71 + 0.82 26.62 + 0.81
22.48 + 1.05 28.16 + 0.97 30.13 + 1.45
22.70 + 1.52 24.61 + 0.88 29.22 + 0.62
23.82 + 0.98 26.12 + 1.61 37.36 + 2.76
21.51 + 1.05 28.48 + 1.72 25.85 + 1.78
19.55 + 0.88 32.37 + 2.54 25.83 + 1.42
19.02 + 0.85 19.94 + 1.09 22.88 + 0.91
18.66 + 0.99 24.42 + 1.55 22.48 + 0.85
20.94 + 1.23 29.41 + 1.43 25.04 + 1.67
LY 294002 (Ly) is a standard PI3K inhibitor (Fig. 2) known to
cause G0/G1 arrest (82.95%). The FACS data clearly suggests
40 that the compounds block the cell cycle at G0/G1phase in A549
cells.
8f
21.22+ 1.27
20.05+ 0.88
17.12+ 0.29
16.83+ 0.33
14.32+ 0.53
18.61+ 0.93
2.84 + 0.27
13.11+ 0.84
12.77+ 0.09
14.81+ 0.55
15.23+ 0.76
4.58 + 0.34
4.15 + 0.82
3.82 + 0.55
3.57 + 0.07
3.26 + 0.42
6.02 + 0.77
17.7 + 1.20
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
2-imino-trifluoromethyl thiazolidinol derivatives induce LC-3
mediated cytoplasmic vacuolation leading to cell death in
45 A549 lung cancer cells
Apart from apoptotic cell death, recent studies have indicated
vital role of autophagic cell death as well as LCꢀ3 mediated
cytoplasmic vacuolation leading to cell death in cancer cell
proliferation7,8. In order to explore the mechanism by which 2ꢀ
50 iminoꢀtrifluoromethyl thiazolidinol derivatives induce cell death
in A549 lung cancer cells, hoescht staining was performed which
indicated that the fluorinated Thiazolidinols had obvious effect
on cell cycle arrest but not on DNA fragmentation. Both the
morphology (Fig. 3a) and subG1% did not provide evidence of
55 apoptosis. Interestingly, compounds 8l, 8s and 8t have exhibited
clear vacuole formation than others. Further, western blot
analysis of microtubule associated protein light chain 3(LCꢀ3)
which is considered as an important marker of vacuole mediated
cell death (Fig. 3a), confirmed LCꢀ3 mediated cell death in cells
60 treated with the compound. LCꢀ3 is expressed as a polypeptide,
and is subsequently cleaved to form LC3ꢀI. LC3ꢀI is converted
to LC3ꢀII via addition of phosphatidylethanolamine (PE) group to
the Cꢀterminus28. Interestingly, we have observed an increase in
the levels of LC3ꢀII in compound treated cells (Fig. 3b) when
65 compared with control untreated cells. Further we also checked
the levels of one key autophagy associated protein Atgꢀ7 in
control and treated A549 cells which showed an increase in the
Atg7 protein levels in treated A549cells compared to control.
3.32 + 0.45
21.66 + 0.63 19.68 + 1.27 25.08 + 0.91
20.85 + 0.88 21.0 + 0.77 25.33 + 1.56
3.57 + 0.38
6.693 + 0.82
21.27 + 0.94 30.74 + 1.65 34.06 + 2.78
20.62 + 1.09 16.83 + 1.47 29.25 + 1.34
6.64 + 0.64
5.92 + 0.22
5.12 + 0.89
4.48 + 0.17
4.03 + 0.34
6.98 + 0.34
6.24 + 0.91
5.84 + 0.76
6.41 + 0.62
5.54 + 0.29
28.65 + 0.89
18.24 + 0.78
15.14 + 0.56
11.44 + 0.83
32.34 + 0.66
11.33 + 0.27
29.1 + 1.76
8t
8u
8v
Ly
19.59 + 1.02 7.12 + 0.08
9.7 + 0.98 20.5 + 1.45
Cell lines were treated with different concentrations of the compounds as
described in Materials and Methods. Cell viability was determined by
MTT assay. Each of the compound was tested in triplicates (n=3). IC50
values are indicated as mean +/ꢀ SD of three independent experiments.
Effect of 2-imino-trifluoromethyl thiazolidinol derivatives on
Cell Cycle distribution
Cell cycle is regulated by 2 classes of genesꢀ the oncogenes and
the tumor suppressors. Dysregulation of these genes results in
loss of control over rate of cell cycle progression which
ultimately ends up in abnormal cell proliferation. Thus, cell cycle
plays a key regulatory role to control abnormal cell proliferation.
Cell cycle arrest by anticancer agents is mainly by control of
5
10 rapid cell proliferation. Thus, we have examined the effect of
these derivatives on cell cycle of A549 cells. Flow cytometry
results indicated that compounds 8l (86.61%), 8r (81.78%), 8s
(79.77%), 8t (75.30%) and 8u (82.31%) show G0/G1 cell cycle
arrest as indicated by increased percentage of cells in this phase
15 when compared to untreated control cells.
70
75
80
85
20
25
30
Fig. 2 Effect on cell cycle. A549 cells were treated with 8l, 8r, 8s, 8t and
8u at 4 ꢁM or LY 294002 (Ly) at 10 ꢁM concentration and incubated for
24h. This was followed by FACS analysis. Compounds exhibited G0/G1
cell cycle arrest as shown by increased percentage of cells in this phase
35 and a decrease in percentage of cells in the G2/M phase. LY 294002 (Ly)
treated A549 cells have shown G0/G1cell cycle arrest as expected.
90 Fig. 3 (a) Effect on cell morphology. Compound treated cells lost their
morphology and have shown vacuole formation (20X)
95
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3

MedChemComm
Page 4 of 8
View Article Online
DOI: 10.1039/C5MD00603A
MEK/ ERK pathway also. This signalling has strong influence on
aggressiveness and rapid proliferation of cancer cells35. A549
cells were treated with the compounds and western blot was
performed using antibody specific to phosphorylated form of
60 MEK and ERK. We checked the levels of pMEK1/2 and
pERK1/2. We observed decrease in the levels of pMEK1/2 and
pERK1/2 revealing that these compounds act on both PI3K
signalling and MEK/ERK signalling as well. It is well reported
that some cues in the cross talks36 which are effectors of PI3K are
65 also effectors of MEK/ERK signalling (Fig.4b).
5
10
70
75
80
Fig. 3 (b). Western blotting indicating the levels of vacuole marker LC3
15 in control and treated cells. During LCꢀ3 mediated cell death, microtubule
associated protein LC3 is cleaved and there is an increase in the levels of
LC3ꢀII. Here the graph shows the relative density of LC3ꢀII against LC3ꢀI
expressed as arbitrary units. X axis represents control and treated samples
while Y axis represents LC3ꢀII/ LC3ꢀI ratio.
20
25
Fig. 4(a) Effect on PI3K/Akt/mTOR pathway.
A549 lung cancer cell line was grown in 60mm dish for 24h. Then cells
85 were incubated with the compounds (Fluorinated Thiazolidinols) at 4ꢁM
or with Ly at 10 ꢁM concentration for 24h. The lysates were harvested,
quantified by Bradford method and were subjected to western blot
analysis. Here, antibodies against PI3K, Akt, pAkt (ser473) and mTOR
were used. X axis represents control and treated samples while Y axis
90 represents the relative protein expression expressed as arbitrary units in
each case i.e., relative density of PI3K, mTOR against βꢀactin and pAkt
relative to total Akt. βꢀactin was used as a loading control. Compounds
reduced the expression of PI3K, Akt, pꢀAkt and mTOR significantly.
30
35 Fig. 3 (c). Western blotting analysis of the key autophagy associated
protein Atg7 in control and treated A549 cells. Autophagy is controlled
by autophagyꢀrelated genes, Atg7 represents a key gene among these.
Here the graph shows the relative density of Atgꢀ7 protein against βꢀactin
expressed as arbitrary units. X axis represents control and treated sample
40 while Y axis represents Atgꢀ7/ βꢀactin ratio.
95
Effect on PI3K/ Akt/mTOR pathway
100
Further, we made an attempt to explore the molecular mechanism
involved in LCꢀ3 induction by these compounds. The stimulation
of class I PI3ꢀKinase activity inhibits vacuolated cell death
45 whereas class III kinases convert phosphatidylinositol (4,5)ꢀ
bisphosphate (PIP2) to phosphatidylinositol (3,4,5)ꢀtrisphosphate
(PIP3) and promote the process29. Recent studies have indicated
possible involvement of PI3K, Akt (PKB) and mTOR in
vacuolation mediated cell death30,31. Therefore, herein we have
50 investigated plausible effect of these compounds on PI3K and its
downstream targets such as Akt and mTOR. We observed down
regulation of PI3K, Akt , phosphorylated form of Akt (Akt Ser
105
Fig. 4(b) Effect on MEK/ERK pathway. A549 lung cancer cell line was
grown in 60mm dish for 24h. Then cells were incubated with the
110 compounds (Fluorinated Thiazolidinols) at 4ꢁM or with Ly at 10 ꢁM
concentration for 24h. The lysates were harvested, quantified by Bradford
method and were subjected to western blot analysis using the indicated
antibodies against pMEK 1/2 and pꢀERK1/2. X axis represents control
473) and mTOR in compound treated A549 cells (Fig. 4a)32ꢀ34
.
Since cancer cell proliferation is controlled by various signalling
55 pathways we checked the effect of these analogues on RAS/
4
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 5 of 8
MedChemComm
View Article Online
DOI: 10.1039/C5MD00603A
and treated samples while
Y
axis represents the relative protein
Lung cancer is the most common cause of cancer related death in
the world with very limited therapeutic options. Therefore, new
therapeutic strategies are urgently needed. Heterogeneity relating
to cancer treatment has been observed in multiple malignant
60 cancers, including lung cancer. Apart from classical cytotoxics
there is a strong demand to identify small molecules that target
growth factor mediated signalling pathways such as PI3K/Akt/
mTOR as well as MEK /ERK signalling.
expression expressed as arbitrary units in each case. Upon compound
treatment we observed a decrease in the levels of pMEK1/2 (relative
density against βꢀactin ) and pERK 1/2 (relative density against total
5
ERK1/2).
Effect on PTEN
PTEN is considered to be an important candidate tumor
suppressor gene that is mutated and lost in many cancers and
functions as a buffer for PI3K. Thus, the loss and mutation
Cancer cells exhibit uncontrolled cell proliferation while antiꢀ
65 cancer drugs show effect on cell viability and cause death of the
cancer cells. In order to examine the cytotoxic activity, the
compounds were tested in A549 (human lung cancer cellsꢀp53
wildꢀtype), MDAꢀMBꢀ231 [(human breast cancer cells (nonꢀ
functional p53)], HeLa (p53 wildꢀtype) and MCFꢀ7 (p53 wildꢀ
70 type) cells. The MTT assay was carried out in order to determine
the cell viability after compound treatment. Out of all the cell
lines tested the compounds were effective in the order of A549
>MDAꢀMBꢀ231> HeLa>MCFꢀ7. Among all the compounds 8l,
8r, 8s, 8t, 8u were found to be effective (Table 1). Further we
75 investigated the possibility of effect of these compounds on cell
cycle as many antiꢀcancer agents function by inducing cell cycle
arrest in cancer cells. Flow cytometry data obtained revealed
G0/G1 cell cycle arrest by these compounds (Fig. 2). We also
examined the morphological features of A549 lung cancer cells
80 during the treatment with these compounds. We found formation
of vacuoles in compound treated cells. These results were further
confirmed by determination of expression levels of microtubule
associated protein LC3. As expected, we observed an increase in
cleaved product of LC3 (i.e. LC3ꢀII). This further confirms the
85 vacuole inducing nature of these compounds (Fig. 3). Also we
observed increased protein expression levels of Atg7, a key
autophagy associated protein in treated A549 cells compared to
control. Earlier in hepatoma cells, by knockdown of the Atg7
expression, it was found that autophagy was inhibited by the
90 reduction of LC3ꢀII expression37.
10 of PTEN in various cancers leads to hyperactive PI3K signaling.
In addition, tumor suppressor protein PTEN (phosphatase and
tensin homologue) is known to promote cell death by inhibiting
PI3K, Akt (PKB) and finally mTOR30,31. Immunocytochemistry
was performed in order to understand the plausible effect of
15 compounds on PTEN expression. We noticed increase in PTEN
expression (PTEN foci in red by Cyꢀ3 staining captured using
confocal microscope) in compound treated A549 cells than
control cells (Fig. 5). This result was confirmed by observing the
PTEN protein expression in compound treated cells. Merged
20 image indicates increased expression of PTEN foci (red) in cells
with nucleus stained in blue colour by DAPI.
25
30
35
40
45
Further, we have examined the effect of these compounds on
key signalling pathways. Phosphoinositide 3ꢀkinase (PI3k)
pathway is an important signalling pathway that integrates
metabolism
and
cell
survival/growth
signals.
The
95 Phosphoinositide 3ꢀkinases (PI3Ks) are a family of proteins
involved in various cellular processes such as regulation of
growth, proliferation, metabolism, glucose homeostasis and
vesicle trafficking. The aberrant function of downstream targets
of PI3K such as Phosphatase and Tensin homolog deleted on
100 chromosome (PTEN), Akt, mTOR were found to be associated
with the development of malignancies. Interestingly, these
compounds have shown decrease in the expression of PI3K, Akt
(ser473) and mTOR with concomitant increase in PTEN tumor
suppressor in A549 cells. We also observed that these compounds
105 decrease the expression of MEK/ ERK. This indicates that these
compounds act as dual inhibitors of PI3K/mTOR38 and also the
MEK/ ERK pathway. Thus, these compounds can be further
assessed in vivo for tumor regression ability. Also based on these
results, these compounds can be considered to have potentiality
110 for further clinical investigations.
50 Fig. 5 Effect on PTEN tumor suppressor. A549 cells were treated with the
compounds at 4 ꢁM and LY 294002 (Ly) at 10 ꢁM concentration and
incubated for 24h. The cells were immunostained with PTEN antibody.
The PTENꢀCy3 staining was captured using confocal microscope.
Red: PTEN, Blue: Nucleus (DAPI), Merge: PTEN+ DAPI.
Conclusion
55 Discussion
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5

MedChemComm
Page 6 of 8
View Article Online
DOI: 10.1039/C5MD00603A
Our study clearly shows that thiazolidinol derivatives suppress
lung cancer growth as indicated by cytotoxicity data. Compounds
also induced cytoplasmic vacuolation in lung cancer cells as
evident by studies on vesicle formation and cleavage of
microtubule associated protein LC3 into LC3ꢀI and LC3ꢀII.
Interestingly, these compounds have the ability to inhibit proteins
of the PI3K/Akt/mTOR pathway and concomitantly activate
PTEN. Now, many antiꢀcancer agents that act on PI3K signalling
or mTOR or PTEN are under clinical trials. Further our study
10 suggests that the identified compound targets PI3K/Akt/mTOR
and the MEK /ERK, the key signalling pathways and has
potential to be used in combinatorial regimen in chemotherapy to
overcome the resistance of lung cancer cells to
chemotherapeutics.
Rodriguez, R. D. Baxter, B. Herle, N. Sach, M. R. Collins, Y.
Ishihara and P. S. Baran, Nature 2012, 492, 95.
55
60
65
70
75
80
85
90
95
12 (a) M. D’hooghe and N. D. Kimpe, Tetrahedron, 2006, 62, 513; (b)
S. Bae, H. G. Hahn, K. D. Nam and H. Mah, J. Comb. Chem., 2005,
7, 7; (c) D.ꢀS. Kim, Y.ꢀM. Joeng, I.ꢀK. Park, H.ꢀG. Hahn, H.ꢀK. Lee,
S.ꢀB. Kwon, J. H. Jeong, S. J. Yang, U. D. Sohn and K C. Park,
Biol. Pharm. Bull., 2007, 30, 180.
5
13 (a) S. P. Singh, S. S. Parmar, K. Raman and V. I. Stenberg, Chem.
Rev., 1981, 81, 175; (b) T. Previtera, M. G. Vigorita, M. Bisila, F.
Orsini, F. Benetolla and G. Bombieri, Eur. J. Med. Chem. 1994, 29,
317; (c) M. V. Diurno, O. Mazzoni, G. Correale and I. G. Monterry,
II Farmaco., 1999, 54, 579.
14 (a) M. G. Vigorita, R. Ottana, F. Monforte, R. Maccari, M. T.
Monforte, A. Trovato, M. F. Taviano, N. Miceli, G. De Luca, S.
Alcaro and F. Ortuso, Bioorg. Med. Chem., 2003, 11, 999–1006; (b)
R. K. Rawal, R. Tripathi, S. B. Katti, C. Pannecougue and E. De
Clerg, Bioorg. Med. Chem., 2007, 15, 1725–1731; (c) R. Ottana, R.
Maccari, R. Ciurleo, M. G. Vigorita, A. M. Panico, V. Cardile, F.
Garufi and S. Ronsisvalle, Bioorg. Med. Chem., 2007, 15, 7618–
7625.
15 Acknowledgements
This work was supported by grants from Council of Scientific
and Industrial research (XII five year plan projectsꢀ DITSF (CSCꢀ
0204) and SMiLE (CSC0111)), Department of Science &
Technology (SR/ FT/CSꢀ031/2010) and Department of
20 Biotechnology (BT/BioꢀCARe/08/511/2010ꢀ11, DBT), India.
T.D. thanks UGC, A.N., Y.R. and S. H. thank CSIR, New Delhi
for the award of fellowship.
15 (a) M. Takagi, K. Ishimitsu and T. Nishibe, WO 0172723 A1, 2001;
[Chem. Abstr. 2001, 135, 288773]; (b) M. Tomizawa, A. Cowan and
J. E. Casida, Toxicol. Appl. Pharmacol., 2001, 177, 77.
16 (a) C. G. Bonde and N. Gaikwad, J. Bioorg. Med. Chem., 2004, 12,
2151; (b) D. M. Vittoria, O. Mazzoni, E. Piscopo, A. Caligmano and
A. Bolognese, J. Med. Chem., 1992, 35, 2910; (c) A. Bodtke, W. D.
Pfeiffer, N. Ahrens and P. Langer, Bioorg. Med. Chem. Lett., 2004,
14, 1509.
References
1
R. AdbulꢀGhani, V. Serra, B. Gyorffy, K. Jurchott, A. Solf, M.
Dietel and R.Schafer, Oncogene, 2006, 25, 1743ꢀ1752.
E. H. Walker, M. E. Pacold, O. Perisic, L. Stephens, P. T. Hawkins,
M. P. Wymann and R. L. Williams, Molecular Cell., 2000, 6, 909–
919.
25
30
35
40
45
50
2
17 G. J. Murphy and J. C. Holder Pharmacol. Sci. 2000, 21, 469ꢀ474.
18 R. B. Lesyk, B. S. Zimenkovsky, D. V. Kaminskyy, A. B.
Kryshchyshyn, D. YaHavryluk, D. V. Atamanyuk, I. Yu Subtel’na
and D. V. Khyluk, Experience of DH LNMU medicinal chemistry
scientific group. 2011, 27, 107–117.
3
(a) A. Akinleye, P. Avvaru, M. Furqan, Y. Song and Delong,J.
Hematology and Oncol., 2013, 6, 88; (b) S. M. Maria, Mol. Cancer
Ther.,2011, 10, 2016.
19 N. Karali, N. Terzioglu and A. Gursoy, Arch. Pharm. Pharm. Med.
Chem. 2002, 8, 374.
4
5
E. Hirsch, E. Ciraolo, A. Ghigo and C. Costa, PharmacolTher.,
2008, 118, 192ꢀ205.
20 D. W. Zaharevitz, S. L. Holbeck, C. Bowerman and P. A. Svetlik, J.
Mol. Graph. Model. 2002, 20, 297.
J. A. Engelman, L. Chen, X. Tan, K. Crosby, A. R. Guimaraes, R.
Upadhyay, M. Maira, K. McNamara, S. A. Perera, Y. Song, L. R.
Chirieac, R. Kaur, A. Lightbown, J. Simendinger, T. Li, R. F.
Padera, C. GarciaꢀEcheverria, R. Weissleder, U. Mahmood, L. C.
Cantlev and K. K. Wong, Nat. Med., 2008, 14, 1351ꢀ1356.
S. S. Yea and D.A. Fruman, Ann N Y Acad sci., 2013, 1280, 15ꢀ18.
P. G. Clarke, Anat embryo (Berl). 1990, 181, 195ꢀ213.
D. Gozuacik and A. Kimchi, Oncogene.2004, 23, 2891ꢀ2906.
(a) G. K. S. Prakash and A. K. Yudin, Chem. Rev. 1997, 97, 757; (b)
B. E. Smart, J. Fluorine Chem. 2001, 109, 3; (c) S. Purser, P. R.
Moore, S. Swallow and V. Gouverneur, Chem. Soc. Rev. 2008, 37,
320
21 K. D. Paull, R. H. Shoemaker, L. Hodes, A. Monks, D. A. Scudiero
and L. Rubinstein, J. Plowman, M. R. Boyd, J. Natl. Cancer Inst.
1989, 81, 1088.
22 S. Kobayashi, S. Hidaka, H. Kawamura, M. Ozaki and Y. Hayase, J.
Antibiot., 1998; 51, 323ꢀ7.
6
7
8
9
23 A. Ino and A. Murabayashi, Tetrahedron, 1999; 55, 10271ꢀ82.
24 R. M. Kumbhare, T. L. Dadmal, T. Anjana Devi, D. Kumar, U. B.
Kosurkar, D. Chowdhury, K. Appalanaidu, Y. K. Rao, M. J.
Ramaiah and M. P. Bhadra, Med. Chem. Commun., 2014, 5, 1744.
100 25 R. M. Kumbhare, T. Dadmal, U. Kosurkar, V. Sridhar and J. V.
Rao, Bioor. & Med. Chem. Lett. 2012, 22, 453–455.
10 (a) K. Müller, C. Faeh and F. Diederich, Science 2007, 317, 1881;
(b) G. K. S. Prakash, P. V. Jog, P. T. D. Batamack and G. A. Olah,
Science 2012, 338, 1324; (c) G. K. S. Prakash, Z. Zhang, F. Wang,
S. Munoz and G. A. Olah, J. Org. Chem. 2013, 78, 3300; (d) W. R.
Dolbier Jr., Chim. Oggi2003, 21, 66.
26 R. M. Kumbhare, T. L. Dadmal, M. J. Ramaiah, K.S.V. Kishore,
S.N.C.V.L. PushpaValli, S. K. Tiwari, K. Appala Naidu, Y. K. Rao
and M. P. Bhadra, Bio. & Med. Chem. Lett., 2015, 25, 654ꢀ658.
105 27 T. Dalmal, K. Appalanaidu, U. B. Kosurkar, N. J. Babu and R. M.
Kumbhare, Eur. J. Org. Chem. 2014, 12, 2468–2479.
11 (a) R. Filler and R. Saha, Fut. Med. Chem. 2009, 1, 777; (b) T.
Furuya, A. S. Kamlet and T. Ritter, Nature, 2011, 473, 470; (c) Y.
Fujiwara, J. A. Dixon, F. O’Hara, E. D. Funder, D. D. Dixon, R. A.
28 N. Mizushima, T. Yoshimorim and B. Levine, Research. Cell. 2010,
140,313ꢀ326.
6
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 7 of 8
MedChemComm
View Article Online
DOI: 10.1039/C5MD00603A
29 E. Ogier Denis and P. Codogno, Biochim. Biophys. Acta., 2003,
1603, 113ꢀ128.
30 S. Arico, A. Petiot, C. Bauvy, P. F. Dubbelhuis and A. J. Meijer, J.
Biol. Chem., 2001, 276, 35243ꢀ35246.
5
31 T. Ueno, W. Sato, Y. Horie, M. Komatsu, I. Tanida, M. Yoshida, S.
Ohshima, T. W. Mak, S. Watanabe and E. Kominami, Autophagy,
2008, 4, 692ꢀ700.
32 R. AbdulꢀGhani, V. Serra, B. Gyorffy, K. Jurchott, A. Solf, M.
Dietel and R. Schafer, Oncogene. 2006, 25, 1743ꢀ1752.
10 33 J. M. Tang, Q. Y. He, R. X. Guo and X. J. Chang, Lung Cancer,
2006, 51, 181ꢀ191.
34 F. Scarlatti, R. Granata, A. J. Meijer and P. Codogno, Cell death
Diff., 2009, 16, 12ꢀ20.
35 A. Kamal, S. Faazil, M. J. Ramaiah, M. Ashraf, M. Balakrishna, S.
15
20
25
N. Pushpavalli, N. Patel and M. PalꢀBhadra, Bioorg Med Chem
Lett., 2013, 23, 5733ꢀ5739.
36 C. Michelle, E. Mendoza, Er. Emrah and B. John, Trends Biochem
Sci., 2011, 36(6), 320–328.
37 Z. Liu, Y. J.Lv, Y. P.Song, X. H. Li, Y. N. Du, C. H. Wang, L.
K.Hu, Oncology Reports . 2012, 28, 985ꢀ991.
38 N. Hay, Cancer Cell, 2005, 8, 179ꢀ183.
30
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 7

MedChemComm
Page 8 of 8
View Article Online
DOI: 10.1039/C5MD00603A
Fluorinated thiazolidinols cause cell death in A549 lung cancer
cells via PI3K/AKT/mTOR and the MAPK /ERK signalling pathways
Ravindra M. Kumbhare,*a Tulshiram L. Dadmal,a,d Dinesh Kumar,b M. Janaki Ramaiah,c Anudeep
Kota, Debabrata chowdhury,b K. Appalanaidu,a Y. Khageswara Rao,a Sayyad Hyder,a T. Anjana
Devi,*b and Manika Pal Bhadra*b
aFluoroorganic Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, 500607, India.
b Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, 500607,
India.
cSchool of Chemical and Biotechnology, SASTRA University, Tirumalaisamudram, Thanjavur 613401,
Tamilnadu, India.
dGovernment of Maharashtra, Ismail Yusuf College, Jogeshwari (East), Mumbai 400060, India
Graphical Abstract
Fluorinated Thiazolidinols act via inhibition of PI3K/Akt/mTOR, and the MEK/ERK
signalling pathways and lead to LC-3 mediated cytoplasmic vacuolation cell death in lung
cancer cells.