ONS-donor ligand based Pt(II) complexes display extremely high anticancer potency through autophagic cell death pathway

Article abstract of DOI:10.1016/j.ejmech.2018.12.052

The current study unveils ONS-donor ligand based Pt(II) complexes with unusual anticancer potency showing higher anticancer effect as compared to cisplatin. This series of Pt(II)(R-salicylaldimine)Cl (C1a-C4a) (R = 5-H, 5-CH₃, F, 3-CH₃

Full text of DOI:10.1016/j.ejmech.2018.12.052

Accepted Manuscript
ONS-donor ligand based Pt(II) complexes display extremely high anticancer potency
through autophagic cell death pathway
Faiz-Ur Rahman, Amjad Ali, Hong-Quan Duong, Inam Ullah Khan, Muhammad
Zeeshan Bhatti, Zhan-Ting Li, Hui Wang, Dan-Wei Zhang
PII:
S0223-5234(18)31095-X
DOI:
https://doi.org/10.1016/j.ejmech.2018.12.052
EJMECH 10986
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 September 2018
Revised Date: 6 December 2018
Accepted Date: 21 December 2018
Please cite this article as: F.-U. Rahman, A. Ali, H.-Q. Duong, I.U. Khan, M.Z. Bhatti, Z.-T. Li, H. Wang,
D.-W. Zhang, ONS-donor ligand based Pt(II) complexes display extremely high anticancer potency
through autophagic cell death pathway, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2018.12.052.
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ACCEPTED MANUSCRIPT
ONS-donor ligand based Pt(II) complexes with high anticancer activity
were prepared. They induced autophagic cell death via LC3-I/LC3-II and
recruitment of LC3B to autophagosomal membranes through activation
of AKT/p21. These complexes showed strong cell growth retardation effect
on E. Coli and resulted in filamentous morphology.

ACCEPTED MANUSCRIPT
ONS-donor ligand based Pt(II) complexes display extremely high
anticancer potency through autophagic cell death pathway
a,b,1
c,1
d,1
e
Faiz-Ur Rahman , Amjad Ali , Hong-Quan Duong , Inam Ullah Khan , Muhammad
c,f
a
a
a
Zeeshan Bhatti , Zhan-Ting Li , Hui Wang * and Dan-Wei Zhang *
a
Center for Supramolecular Material and Catalysis, Department of Chemistry, Shanghai University, Shanghai
2
00444, China.
b
Department of Chemistry, Fudan University, 2005 Songhu Road, Shanghai 200438, China
c
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences, School of Life Sciences,
East China Normal University, 500 Dongchuan Road, Shanghai 200241, China
d
Institute of Research and Development, Duy Tan University, K7/25 Quang Trung, Danang 57000, Vietnam.
e
Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences,
Fudan University, 220 Handan Road, Shanghai 200433, China.
f
Department of Biological Sciences, National University of Medical Sciences, Rawalpindi, 46000, Pakistan.
*Corresponding Authors E-mail: zhangdw@fudan.edu.cn (D.-W. Zhang), wanghui@fudan.edu.cn
(
H. Wang)
Tel.: +86 21 65643576
Abstract
The current study unveils ONS-donor ligand based Pt(II) complexes with unusual anticancer
potency showing higher anticancer effect as compared to cisplatin. This series of Pt(II)(R-
salicylaldimine)Cl (C1a-C4a) (R = 5-H, 5-CH , F, 3-CH O) complexes were prepared in single
3
3
step in good isolated yields from commercially avialable materials. The chloride ancillary ligand
of “a” series (C1a-C4a) was replaced with 4-picoline and “b” series of four complexes Pt(II)(R-
1
F. -U. Rahman, A. Ali and H.-Q. Duong have equal contribution in this work

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4 3 3
salicylaldimine)(4-picoline)BF (C1b-C4b) (R = 5-H, 5-CH , F, 3-CH O) was obtained. All
these complexes were characterized by different structure elucidation techniques. Among these,
the structures of C1a, C2a, C2b and C3b were determined in solid state by single crystal X-ray
analysis. We found quick aquation of “a” series of complexes in DMSO/water mixture that was
1
well investigated by H NMNR, LCMS and ESI-MS, while “b” series of these complexes was
1
quite stable over a month as described by the H NMNR in DMSO/D O mixture. This ONS-
2
donor ligand based class of Pt(II) complexes showed unusual anticancer potency in non-small
cell lung cancer A549, colorectal cancer HT-29 and triple negative breast cancer MDA-MB-231
cells. These Pt(II) complexes induced PARP cleavage and significantly inhibited colony
formation ability of cancer cells. Mechanistically, we found reduced aggressive growth of cancer
cells by the induction of autophagic cell death via LC3-I/LC3-II expression and recruitment of
LC3B to autophagosomal membrane. These complexes induced p21 expression, that suggested
their potentials to suppress cell cycle progression. Significant activation of caspase3/7-dependent
apoptotic signaling was observed in cancer cells treated with these Pt(II) complexes.
Morphological changes of cancer cells suggested their potentials to modulate epithelial-
mesenchymal-transition (EMT) like features of cancer cells. Gel electrophoresis study revealed
their interaction with plasmid DNA. Similarly, strong growth retardation effect and filamentous
morphology was observed in Escherichia coli (E. coli). These ONS-donor Pt(II) complexes
possessed strong anticancer effect in multiple human cancer cells via activation of multiple
pathways for apoptotic and autophagic cell death.
Key words
ONS-donor Pt(II) complexes, In vitro cytotoxicity, Caspase 3/7 activation, Autophagy, E. Coli
growth.

ACCEPTED MANUSCRIPT
1
. Introduction
Platinum-based complexes have been approved as anticancer drugs (Fig. 1). According to an
estimation, more than 50% of cancer treatments comprised of these platinum based drugs [1-3].
The major limitations associated with these drugs were their poor activity in certain types of
cancers, inactivation by intracellular biomolecules, side effects and timely acquired or intrinsic
resistance in cancer patients [4-8]. These limitations increase the need for developing new
platinum-based anticancer agents with new assemblies which could increase the potency and
minimize the other related limitations [9]. In designing of new platinum-based chemotherapeutic
agents, emphasis was placed on the diversity of the coordinated ligands and new agents were
prepared with different mode of action and favourable cytotoxicity in cancer cells as compared to
the classical cisplatin [1, 10-15]. Though a number of, may be in thousands cytotoxic platinum
complexes were reported but most of them were abandoned during initial clinical trials due to
their inappropriate clinical effects [2, 16]. Struggles to develop novel platinum based
chemotherapeutic drugs were continued for example Lippard and co-worker recently discovered
a new class of monofunctional antineoplastic agents synthesized from cisplatin, finding a way to
become platinum based anticancer drug candidates those showed better activities compared to
the parent cisplatin [17-21].

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Fig. 1 Clinically administered (approved) platinum based anticancer drugs; cisplatin, carboplatin and oxaliplatin are
globally approved while nedaplatin, lobaplatin and heptaplatin are regionally approved in China, South Korea and
Japan respectively.
Non-small cell lung carcinoma (NSCLC), colorectal carcinoma (CRC) and triple negative breast
cancer (TNBC) are the most common and aggressive cancers develop in human body [22, 23].
Triple negative breast cancer has ability to metastasize to secondary parts of the body such as
lungs, brain and colon. TNBC is a highly metastatic disease and cell from these patients often
show very aggressive nature in culture. Mutations in these tumors make them highly aggressive,
which is characterized by uncontrolled cell growth. Activation of growth factor receptor
signaling, amplification of tumor promoting proteins and inhibition of tumor suppressor genes
functions play a key role in cancer cell growth, survival and proliferation [24]. One of the
hallmarks of cancer cells is to show resistance against chemotherapeutic agents due to activation
of survival signaling, which in turn decreases the survival rate of cancer patients and contribute
to poor prognosis[25]. Cancer cells often show less induction of apoptosis due to constant
activation of multiple oncogenic signaling. Cancer cells often follow apoptotic signaling for cell
death but in most cases, cell death also occurs through induction of autophagy[26, 27].
Autophagy is the alternative mechanism for cancer cell death, in which autophagosome play
critical role in cell death. Activation of LC3-I and LC3-II proteins represents activation of
autophagy mediated cell death[26].
Cancer patients showing resistance to conventional chemotherapeutic or other targeted therapies
such as endocrine therapy and immunotherapy finally succumb to metastatic disease, which is
one of the leading cause of cancer related mortality [28]. Currently, several anticancer agents are
in clinical trials due to their cytotoxicity towards cancer cells. However, in most cases these
agents display several side effects and therefore most of the clinical trials failed [2, 29, 30].

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Therefore, identification and characterization of novel chemotherapeutic agents are considered
important in clinical trials for more effective treatment and prolong survival of cancer patients.
The final goal of these treatments is to reduce tumor burden and prevent recurrence and relapse.
An important property of S-donor ligand is the incorporation of chirality on sulfur center after
coordination with metal [31-33]. The neighboring group attached to sulfur affect the R or S
stereoisomer formation on sulfur center. If the attached alkyl group is achiral like phenyl and
methyl (methyl phenyl thioether) there is always expected a racemic mixture of R and S
stereoisomers [34]. The presence of a neighboring chiral center attached to sulfur increase the
particular anti or syn stereoisomers formation after coordination with metal [33]. This property
of chirality on sulfur on a S-donor ligand was reported in a number of important metal catalyzed
chiral catalysis [35]. Similarly, the already chiral sulfoxide ligand were also reported in some
studies that retained its particular chirality after coordination with metal and functioned as chiral
catalyst [36, 37]. Salicylaldimine based related ligand bearing such chirality or incorporation of
chirality on S-donor atom after coordination with metal was observed but up to our knowledge it
was not discussed previously [25, 38-41].
Salicylaldimine chelating ligands are important assemblies for the synthesis of a number
of metal complexes, some of them were studied for their biological properties [42-45]. Platinum
complexes of salicylaldimine ligands were mostly reported for their photo physical properties
[46-49]. There are limited examples of platinum complexes based on salicylaldimine ligands
with anticancer effect [50]. Heteroliptic Pt(II) complexes of salicylaldimine and cyclooctene co-
ligands were reported for better anticancer effect in brain cancer [51]. We recently dedicated our
efforts to the synthesis and characterization of salicylaldimine based mixed ligands Pt(II)
complexes with anticancer activities, genes activation and effective bacterial growth retardation

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[52-57]. We established easy, productive and reproducible synthetic methodologies for these
complexes from commercially available raw materials. In search of potent Pt(II) anticancer
agents we continued our efforts to explore further structurally modified salicylaldimine based
complexes and their anticancer properties.
In the current study we synthesized ONS-donor salicylaldimine ligand based Pt(II) complexes
and structurally characterized them in details. Further, we have identified these Pt(II) complexes
for their potent in vitro anticancer effect in multiple human cancer cells. Several mechanistic
assays were conducted to investigate and compare their potency with market available anticancer
drug cisplatin. They induced significant cancer cell death via activation of autophagy and
caspase3/7-dependent signalling. Superior growth retardation effect and filamentous morphology
was observed in Escherichia coli (E. coli) as compared to cisplatin.
2
. Materials and methods
2
.1. General experimental and materials (chemistry)
All analytical grade solvents and reagents were purchased from commercial sources and used
without further purification unless otherwise mentioned. K PtCl was purchased from Energy
2
4
1
19
13
Chemical Company Shanghai. H, F and C NMR analyses were conducted on bruker
AVANCE III HD 400 MHz spectrophotometer at room temperature (298 K) in CDCl , D O or
3
2
DMSO-d . LC-MS and HR-ESI MS analyses were performed on Agilent LC coupled with HR-
6
MS machine Bruker micrOTOF II. X-ray data were collected on a Bruker APEX DUO
diffractometer with graphite-monochromatized Mo Kα radiation (λ= 0.71073 Å). All the
structures were solved by Direct Method of SHELXS-97 and refined by full-matrix least squares
techniques using the SHELXL-97 program within WINGX.[58, 59] Non-hydrogen atoms of the

ACCEPTED MANUSCRIPT
crystallized compounds were refined with anisotropic temperature parameters. The hydrogen
atoms attached to carbons were generated geometrically. Crystallographic data for C1a, C2a,
C2b and C3b, CCDC 1482361-1482364, can be obtained from Cambridge Crystallographic
Data Center 12 Union Road, Cambridge CB2 1EZ, UK; tel: +44-1223336408; fax: +44-
1
223336003; e-mail: deposit@ccdc.cam.ac.uk.
2.1.1. General procedure for the synthesis of ligands (L1 - L4)
1
mmol of particular salicylaldehyde and 1 mmol of 2-(methylthio)aniline were taken in 10 mL
of CH Cl /MeOH (1:1) mixture. The reaction mixture was stirred at 50 °C for 10 h. After
2
2
completion the mixture was dried with anhydrous sodium sulfate and vacuum evaporated. L1–
L4 were recrystallized from cold ethanol in freezer and characterized as below. Spectroscopic
characterization data were given below and original spectra were given in the ESI.
1
L1[25], Yield and spectroscopic analysis results were similar as reported previously. H NMR
(400 MHz, CDCl3) δ 8.6 (s, 1H), 7.47 – 7.37 (m, 2H), 7.32 – 7.27 (m, 2H), 7.24 – 7.16 (m, 2H),
7
.1 (d, J = 8.2 Hz, 1H), 7.0 (t, J = 7.4 Hz, 1H), 2.5 (s, 3H).
1
L2, 63% isolated yield, yellow solid. H NMR (400 MHz, DMSO-d ) δ 12.7 (s, 1H), 8.9 (s, 1H),
6
7
1
1
.4 (s, 1H), 7.4 (d, J = 7.7 Hz, 1H), 7.34 – 7.26 (m, 2H), 7.26 – 7.18 (m, 2H), 6.9 (d, J = 8.4 Hz,
1
3
6
H), 2.4 (s, 3H), 2.3 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 163.1, 158.5, 145.0, 134.9, 134.7,
33.0, 128.1, 128.0, 125.6, 125.0, 119.4, 118.0, 117.0, 20.4, 14.2. HRMS (ESI): Calcd for
+
C H NOSNa 280.0772: [M+Na] , Found: 280.0767.
14
13

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1
L3, 78% isolated yield, yellowish orange solid. H NMR (400 MHz, DMSO-d
6
) δ 12.7 (s, 1H),
8
.9 (s, 1H), 7.5 (dd, J = 8.9, 3.2 Hz, 1H), 7.4 (d, J = 7.8 Hz, 1H), 7.35 – 7.20 (m, 4H), 7.0 (dd, J
1
9
13
=
9.0, 4.5 Hz, 1H), 2.5 (s, 3H). F NMR (376 MHz, DMSO-d ) δ -125.1 - -125.2 (m). C NMR
6
(
100 MHz, DMSO-d ) δ 161.9, 161.9, 156.9, 155.3 (d, J = 234.5 Hz), 144.7, 135.1, 128.4, 125.4
6
(d, J = 54.5 Hz), 120.9 (d, J = 23.6 Hz), 119.9 (d, J = 7.7 Hz), 118.5 (d, J = 7.7 Hz), 118.0, 117.7
+
(
d, J = 23.4 Hz), 14.2. HRMS (ESI): Calcd for C H NFOSNa 284.0521: [M+Na] , Found:
1
4
12
2
84.0528
1
L4, 87% isolated yield, red solid. H NMR (400 MHz, DMSO-d ) δ 13.1 (s, 1H), 8.9 (s, 1H), 7.4
6
(d, J = 7.8 Hz, 1H), 7.34 – 7.29 (m, 2H), 7.26 – 7.20 (m, 2H), 7.1 (d, J = 7.9 Hz, 1H), 6.9 (t, J =
1
3
7
1
.9 Hz, 1H), 3.8 (s, 3H), 2.4 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 163.4, 150.9, 148.4,
6
44.7, 134.9, 128.2, 125.6, 125.1, 124.6, 119.6, 119.1, 118.0, 116.2, 56.3, 14.2. HRMS (ESI):
+
Calcd for C15
H15NO
2
SNa 296.0721: [M+Na] , Found: 296.0704.
2.1.2. General procedure for the synthesis of C1a‒C4a
K PtCl (0.05 mmol) was taken in 1 mL of DMSO and 5 mL of methanol and stirred vigorously
2
4
to dissolve the solid at room temperature. Particular ligand (L1 - L4), 0.05 mmol and NaOAc
0.05 mmol) were dissolved in 15 mL of MeOH in a 50 mL round bottom flask and heated to
reflux. K PtCl solution prepared previously was added to it in one portion and the mixture was
(
2
4
stirred at reflux for 24 h. After completion, the mixture was cooled to rt and volatiles were
evaporated to minimum volume (approx. 2 mL). 10 mL of water was added to the flask with
constant stirring. The yellow to orange precipitate formed was filtered, washed with water and

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2 2
dried in air. The solid complex (C1a-C4a) was further recrystallized form CH Cl -n-hexane and
characterized as below. Spectroscopic characterization data were given below while the original
spectra were given in the ESI.
1
C1a, 78% yield, yellow solid. H NMR (400 MHz, DMSO-d ) δ 9.6 (s, J
= 64 Hz 1H), 8.4 (d,
6
Pt-H
J = 8.6 Hz, 1H), 8.1 (d, J = 7.7 Hz, 1H), 7.9 (d, J = 6.9 Hz, 1H), 7.6 (t, J = 7.6 Hz, 2H), 7.48 (t, J
1
3
=
7.5 Hz, 1H), 7.0 (d, J = 8.6 Hz, 1H), 6.8 (t, J = 7.4 Hz, 1H), 2.9 (s, 3H). C NMR (100 MHz,
DMSO-d
6
) δ 163.0, 153.5, 153.1, 137.2, 136.4, 132.0, 131.9, 129.8, 129.2, 121.1, 121.0, 119.0,
+
1
17.3, 28.4. HRMS (ESI): Calcd for C14
H
12ClNOPtSNa 494.9874: [M+Na] , Found: 494.9869.
1
C2a, 81% isolated yield, yellow solid. H NMR (400 MHz, DMSO-d ) δ 9.6 (s, J
= 64 Hz,
6
Pt-H
1
7
1
2
H), 8.4 (d, J = 8.5 Hz, 1H), 8.1 (d, J = 7.8 Hz, 1H), 7.6 (s, 1H), 7.6 (t, J = 7.3 Hz, 1H), 7.51 –
1
3
6
.38 (m, 2H), 6.9 (d, J = 8.7 Hz, 1H), 2.9 (s, 3H), 2.3 (s, 3H). C NMR (100 MHz, DMSO-d ) δ
61.5, 153.1, 153.0, 139.0, 134.8, 132.0, 131.9, 129.6, 129.1, 125.5, 120.9, 120.6, 118.8, 28.3,
+
0.1. HRMS (ESI): Calcd for C H ClNOPtSNa 509.0030: [M+Na] , Found: 509.0020.
1
5
14
1
C3a, 83% yield, orange solid. H NMR (400 MHz, DMSO-d ) δ 9.66 (s, J
= 68 Hz, 1H), 8.40
6
Pt-H
(d, J = 8.5 Hz, 1H), 8.10 (d, J = 7.7 Hz, 1H), 7.68 (d, J = 9.6 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H),

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1
9
7
6
.56 – 7.46 (m, 2H), 7.00 (dd, J = 9.3, 4.6 Hz, 1H), 2.90 (s, 3H). F NMR (376 MHz, DMSO-d )
1
3
δ -129.0 - 129.2 (m). C NMR (100 MHz, DMSO-d ) δ 159.8, 153.6 (d, J = 231 Hz), 152.8,
6
1
1
52.8, 152.8, 132.1, 132.0, 129.7 (d, J = 58.3 Hz), 125.7 (d, J = 25.0 Hz), 122.6 (d, J = 7.9 Hz),
19.8 (d, J = 3.0 Hz), 118.9, 118.3 (d, J = 22.6 Hz), 28.3. HRMS (ESI): (ESI): Calcd for
+
C H ClFNOPtSNa 512.9779: [M+Na] , Found: 512.9769.
14
11
1
C4a, 87% isolated yield, reddish orange solid. H NMR (400 MHz, DMSO-d ) δ 9.6 (s, J
=
6
Pt-H
6
2
0 Hz, 1H), 8.4 (d, J = 8.6 Hz, 1H), 8.1 (d, J = 7.8 Hz, 1H), 7.6 (t, J = 7.7 Hz, 1H), 7.5 – 7.4 (m,
1
3
H), 7.2 (d, J = 7.3 Hz, 1H), 6.7 (t, J = 7.9 Hz, 1H), 3.8 (s, 3H), 2.9 (s, 3H). C NMR (100 MHz,
DMSO-d ) δ 154.7, 153.2, 153.1, 151.0, 132.0, 131.9, 129.6, 129.2, 127.3, 121.0, 118.9, 116.5,
6
+
1
5
16.4, 56.2, 28.4. HRMS (ESI): (ESI): Calcd for C H ClNO PtSNa 524.9979: [M+Na] , Found:
15
14
2
24.9953.
2.1.3. General procedure for the synthesis of C1b‒C4b
A mixture of 0.05 mmol of particular complex (C1a to C4a) and 0.05 mmol of AgBF was
4
stirred in 10 mL CH CN at 60 °C for 2 h. Solid AgCl precipitated was filtered from the mixture
3
through celite. The yellowish filtrate collected was evaporated to dryness and a yellow solid was
obtained. It was added with 10 mL of CH
was stirred at rt for 24 h and evaporated. The yellow to orange solid recovered was dissolved in
CH Cl and passed through a 2 × 5 cm silica column eluted with 1 % CH OH-CH Cl . After
evaporation of the volatile a yellow to orange solid for each complex (C1b-C4b) was recovered.
2 2
Cl and excess (2 drops) of 4-picoline. This mixture
2
2
3
2
2

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2 2
The solid was further recrystallized form CH Cl -n-hexane and characterized. Spectroscopic
characterization data were given below while the original spectra were given in the ESI.
1
C1b, 73% isolated yield, orange solid. H NMR (400 MHz, DMSO-d
6
) δ 9.5 (s, 1H), 8.8 (d, J =
6
.6 Hz, 2H), 8.5 (d, J = 8.5 Hz, 1H), 8.1 (d, J = 7.9 Hz, 1H), 8.0 (d, J = 8.1 Hz, 1H), 7.7 – 7.65
(m, 1H), 7.64 – 7.6 (m, 3H), 7.5 (t, J = 7.5 Hz, 1H), 7.0 (d, J = 8.5 Hz, 1H), 6.9 (t, J = 7.0 Hz,
1
9
13
1
H), 2.8 (s, 3H), 2.5 (s, 3H). F NMR (376 MHz, DMSO-d ) δ -148.2, -148.3. C NMR (100
6
MHz, DMSO-d ) δ 162.7, 156.3, 153.4, 152.8, 152.0, 138.1, 136.9, 132.6, 132.3, 130.3, 128.3,
6
1
27.3, 121.3, 120.6, 119.4, 118.3, 28.7, 21.2. HRMS (ESI): Calcd for C H N OPtS 530.0866:
20 19 2
+
[
4
M-BF ] , Found: 530.0870.
1
C2b, 79% isolated yield, yellowish organge solid. H NMR (400 MHz, DMSO-d ) δ 9.4 (s, 1H),
6
8
.8 (d, J = 6.5 Hz, 2H), 8.5 (d, J = 8.6 Hz, 1H), 8.1 (d, J = 7.8 Hz, 1H), 7.7 (s, 1H), 7.7 (t, J = 7.4
Hz, 1H), 7.6 (d, J = 6.3 Hz, 2H), 7.5 (t, J = 7.5 Hz, 1H), 7.5 (d, J = 8.8 Hz, 1H), 6.9 (d, J = 8.7
1
9
6
Hz, 1H), 2.8 (s, 3H), 2.5 (s, 3H), 2.3 (s, 3H). F NMR (376 MHz, DMSO-d ) δ -148.2, -148.3.
1
3
C NMR (100 MHz, DMSO-d ) δ 161.2, 155.8, 153.4, 152.9, 152.0, 139.8, 135.4, 132.6, 132.3,
6
1
30.1, 128.3, 127.2, 126.6, 120.8, 120.4, 119.2, 28.6, 21.2, 20.1. HRMS (ESI): Calcd for
+
C H N OPtS 544.1022: [M-BF ] , Found: 544.1034.
21
21
2
4

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1
C3b, 68% yield, yellow solid. H NMR (400 MHz, DMSO-d ) δ 9.5 (s, 1H), 8.8 (d, J = 6.5 Hz,
6
2
H), 8.4 (d, J = 8.6 Hz, 1H), 8.2 (d, J = 7.9 Hz, 1H), 7.8 (dd, J = 9.5, 3.2 Hz, 1H), 7.7 (t, J = 7.9
1
9
Hz, 1H), 7.64 – 7.50 (m, 4H), 7.0 (dd, J = 9.4, 4.7 Hz, 1H), 2.8 (s, 3H), 2.5 (s, 3H). F NMR
1
3
(376 MHz, DMSO-d
6 6
) δ -127.6 – -127.8 (m), -148.2, -148.3. C NMR (100 MHz, DMSO-d ) δ
1
59.6, 155.7, 154 (d, J = 232.0 Hz), 153.5, 152.8, 152.0, 132.6, 132.4, 130.5, 128.3, 127.5, 126.5
(d, J = 25.0 Hz), 122.3 (d, J = 7.9 Hz), 120.2 (d, J = 8.8 Hz), 119.3, 118.9 (d, J = 22.8 Hz), 28.7,
+
2
1.2. HRMS (ESI): Calcd for C H FN OPtS 548.0772: [M-BF ] , Found: 548.0772.
2
0
18
2
4
1
C4b, 82% isolated yield, orange solid. H NMR (400 MHz, DMSO-d
6
) δ 9.4 (s, 1H), 8.8 (d, J =
.1 Hz, 2H), 8.4 (d, J = 8.6 Hz, 1H), 8.1 (d, J = 7.8 Hz, 1H), 7.6 (t, J = 7.7 Hz, 1H), 7.6 (d, J =
.9 Hz, 2H), 7.5 (t, J = 7.5 Hz, 1H), 7.4 (d, J = 8.2 Hz, 1H), 7.2 (d, J = 7.3 Hz, 1H), 6.8 (t, J =
6
5
7
1
1
1
9
.9 Hz, 1H), 3.8 (s, 3H), 2.8 (s, 3H), 2.5 (s, 3H). F NMR (376 MHz, DMSO-d ) δ -148.2, -
6
1
3
48.3. C NMR (100 MHz, DMSO-d ) δ 155.8, 154.0, 153.4, 152.8, 151.9, 150.3, 132.6, 132.4,
6
30.2, 128.2, 127.6, 127.1, 121.2, 119.2, 117.5, 117.5, 56.6, 28.8, 21.2. HRMS (ESI): Calcd for
+
C
21
H
21
N
2
O
2
PtS 560.0971: [M-BF
4
] , Found: 560.0975.

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2
.2. General experimental (Biology)
2.2.1. Cell culture and reagents
Non-small cell lung cancer (A549), colorectal cancer (HT-29) and triple negative breast cancer
(MDA-MB-231) cells were purchased from American Type Culture Collection (ATCC,
’
Manassas, VA). HT-29 cells were cultured in McCoy s 5A supplemented with 10% heat-
inactivated fetal bovine serum (HI-FBS; Gibco, Life Technologies, Naerum, Denmark) and 100
units/mL penicillin/streptomycin. MDA-MB-231 and A549 cells were cultured in DMEM
supplemented with 10% HI-FBS, 1% glutamine and 100 units/mL penicillin/streptomycin.
2.2.2. MTS assay
TM
HT-29, MDA-MB-231 or A549 Cells counted by the TC20 Automated Cell Counter (Bio-Rad,
Pleasanton, CA, USA) were plated in 96-well flat bottom plates at a density of 2000 cells per
well in triplicate and then treated with various concentrations (0, 1, 3, 5 and/or 10 µM) of each
complexes (C1a, C1b, C2a, C2b, C3a, C3b, C4a and/or C4b) or cisplatin (10 µM) for 72 h.
Cell viability was determined using the MTS assay reagent (CellTiter 96 AQueous one Solution
Cell proliferation Assay; Promega, Madison, WI, USA) according to the manufacturer’s protocol.
2
The absorbance was measured at 490 nm using a Wallac Victor 1420 Multilabel counter (Perkin
Elmer, Wellesley, MA). Measurements were performed and the concentration required for a 50%
inhibition of viability (IC ) was determined graphically. Standard Graph was plotted by taking
5
0
concentration of the complexes and relative cell viability. Percentage of cell survival in the
untreated condition was designated as 100%. Relative cell viability = (experimental absorbance-
background absorbance)/(absorbance of untreated control-background absorbance) x 100%.

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2.2.3. Clonogenic assay
HT-29 and A549 cells were seeded in 60 cm dishes at a density of 3000 cells per dish in
duplicate. 24 h after plating, various concentrations (0, 0.1, and/or 0.3 µM) of each complex
(C1b, C2b, C3b and/or C4b) or cisplatin (0.25 µM) were added to each dish. After treatment for
2
4 h, cells were washed by PBS and further incubated for 15 days. Cells were subsequently
stained with 0.5% crystal violet in 25% methanol-containing PBS. Colonies were examined
under a light microscope and counted after capturing images, as described [60]
2.2.4. Western blot analysis
HT-29 or A549 cells were grown to ~70% confluency and reagents were added as indicated
concentrations. Cells were lysed in a buffer containing 20 mM Tris-HCl, 0.5 M NaCl, Triton X-
1
00, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycophosphate, 10 mM NaF, 300 µM Na VO , 1
3 4
mM Benzamidine, 2 µM PMSF, and 1 mM DTT. The protein concentration was determined
using the BCA protein assay (Thermo Scientific, Rockford, IL, USA). Proteins were separated
on SDS-PAGE, transferred on to PVDF membrane, blocked in 5% nonfat milk and probed with
the following antibodies: phospho-ERK1/2 (Y202/T204), phospho-AKT (S473), PARP, GAPDH
and AKT (Cell Signaling Technology, Boston, MA, USA), ERK1/2 and p21 (Santa Cruz
Biotechnology, Santa Cruz, CA, USA) and LC3B (Thermo Scientific). Then, the membranes
were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham
Biosciences, GE Healthcare, Piscataway, NJ, USA) for 1.5 h. Signals were revealed by
’
chemiluminescence (Thermo Scientific) according to the manufacturer s recommendation and
exposed with X-ray film (Thermo Scientific).

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2.2.5. Caspase-3/7 activity assay
Caspase-3/7 activities were quantified using the Caspase-3/7 Glo Assay (Promega) according to
’
the manufacturer s protocol. HT-29 or A549 Cells were treated with various concentrations (0, 1
and/or 3 µM) of each complex (C1b, C2b, C3b and/or C4b) or cisplatin (10 µM) for 24 h and
Caspase-3/7 activities were quantified from cell lysates. Luminescence was measured at 490 nm
2
using Wallac Victor 1420 Multilabel counter (Perkin Elmer). Luminescence values in vehicle-
treated control samples were set to 1 and values obtained in other experimental conditions were
relative to that.
2.2.6. Immunofluorescences
5
A549 cells (1.5 × 10 per well of six-well plates) were seeded onto cover slip. Cells were
untreated or treated with C2b (3 µM) or cisplatin (10 µM) for 18 h. After then, cells were rinsed
with ice-cold PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature
followed by permeabilization with 0.25% Triton X-100 for 10 minutes at room temperature.
Cells were then incubated with 1% BSA for 45 minutes and subsequently incubated with primary
antibodies for overnight at 4 °C followed by 1 h of incubation at room temperature with FITC-
conjugated secondary antibodies. Images were acquired with the confocal system of Leica SP5
inverted microscope (Leica Microsystems, Wetzlar, Germany).
2.2.7. Immunofluorescences
’
Statistical comparisons were made using the two-tailed student s t-test where appropriate. Results
were considered significant in all experiments at * means P < 0.05, ** means P < 0.01 and ***
means P< 0.001. Data were expressed as the mean ± SD of the mean.

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3
. Results
3
.1. Chemistry
3.1.1. Synthesis and structure elucidation
The new series of Pt(II) complexes of ONS-donor substituted salicylaldimine ligands viz
Pt(II)(salicylaldimine)Cl (C1a), Pt(II)(5-methylsalicylaldimine)Cl (C2a), Pt(II)(5-
fluorosalicylaldimine)Cl (C3a) and Pt(II)(3-methoxysalicylaldimine)Cl (C4a) were prepared in
excellent isolated yields (Scheme 1). Chloride ancillary ligand in C1a-C4a was further
exchanged with 4-picoline. C1a-C4a was treated with an equivalent amount of AgBF
4
and
excess 4-picoline subsequently and new series of four complexes i.e.
a
Pt(II)(salicylaldimine)(picoline)BF (C1b), Pt(II)(5-methylsalicylaldimine)(picoline)BF (C2b),
4
4
Pt(II)(5-fluorosalicylaldimine)(picoline)BF4
(C3b)
and
Pt(II)(3-
methoxysalicylaldimine)(picoline)BF (C4b) were obtained in excellent isolated yields (Scheme
4
1
19
13
1
). The structures of all ligands and complexes were characterized by H, F and C NMR
spectroscopy and high-resolution electrospray ionization (HR-ESI) mass spectrometry (ESI Fig.
S9-48). Structures of C1a, C2a, C2b and C3b were determined by single crystal X-ray analysis.
In case of each ligand (L1-L4), we can observe the phenolic OH proton chemical shift in the
most downfield region 12.7-13.12 ppm for L1-L4. The imine proton chemical shifts for these
ligands were also observed in nearly similar region (8.6-8.9 ppm). The other prominent peak of
methyl attached to sulfur atom was similar in all ligand and observed around 2.45 ppm. When
each ligand was reacted with K PtCl4 and C1a-C4a were prepared we observed the
2
disappearance of phenolic “OH” proton for each ligand, showing the covalent bond formation
between Pt(II) and O atoms. similarly clear changes were observed in the imine proton chemical
shift which was shifted about 1 ppm up-field in each complex as compared to the respective

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ligand. The CH
3
protons chemical shift of the methyl group attached to S atom was also shifted
about 0.4 ppm downfield as compared to the chemical shift observed in case of each free ligand
L1-L4), showing the coordination of Pt(II) with S atom. The other aromatic protons were also
(
shifted little up or down field. In case of “b” series of complexes we found three new chemical
shifts two doublet peaks in the aromatic regions around 8.82 and 7.62 ppm each for two protons
of the pyridine part of 4-picoline and one singlet peak around 2.48 ppm for three proton of the
methyl group of 4-picoline. This showed the coordination of one 4-picoline as co-ligand to Pt(II)
1
3
center. C NMR peaks of each ligand and complex showed similar trend. The imine carbon
peak was observed the most downfield for each ligand and Pt(II) complex. Each carbon chemical
shift was shifted little up or down field in “a” series of complexes as compared to the respective
ligand and all these peaks were found similar in “b” series of complexes. In “b” series three new
signals in the aromatic region were observed, assigned to pyridine part and one in the up field
region assigned to methyl carbon of the co-ligand 4-picoline.
Each symmetrical salicylaldimine ligand (L1-L4) coordinate with Pt metal and gave a racemic
mixture of asymmetric complex (C1a-C4a) bearing S-donor atom as stereo-center. Both these
stereo-isomers can be observed in the single crystal packing of C1a, C2a, C2b and C3b (Fig. 3).
The selectivity for a particular stereoisomer is difficult to optimize and similarly isolation of
these isomers may also be difficult.

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MeOH/CH Cl
2
2
O
R
N
+
R
H N
2
S
OH
50 °C, 10 h
OH
L1 - L4
S
K PtCl , NaOAc,
2
4
1
. AgBF , MeCN, 60 °C, 2 h
R
N
4
MeOH/DMSO
reflux, 24 h
S
Pt
2
. CH Cl , picoline, rt, 24 h
O
2
2
Cl
C1a-C4a
L1: R = H, 57%
L2: R = 5-Me, 63%
L3: R = 5-F, 78%
C1a: R = H, 78%
C2a: R = 5-Me, 81%
C3a: R = 5-F, 83%
N
R
Pt
S
O
.BF4
L4: R = 3-CH O, 87%
C4a: R = 3-CH O, 87%
3
3
N
C1b: R = H, 73%
C2b: R = 5-Me, 79%
C3b: R = 5-F, 68%
C4b: R = 3-CH O, 82%
3
C1b-C4b
Scheme 1. Synthetic route for L1-L4, C1a-C4a and C1b-C4b
Fig. 2. Single crystal plot of (A) C1a, (B) C2a, (C) C2b and (D) C3b.
3.1.2. Single crystal description of C1a, C2a, C2b and C3b
X-ray quality crystals of C1a, C2a, C2b and C3b were grown from CH Cl –n-hexane solution
2
2
of these complexes. The ORTEP plot of these crystals was displayed in Fig. 2. The crystal

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structure refinement parameters for these complexes are summarized in ESI table S1 and
structure plots of these complexes were displayed in ESI Fig S5-8. All these complexes
crystalized in monoclinic crystal systems where Pt adopts square planner symmetry. The single
crystal packing of each complex was consisted of racemic mixture of R and S stereoisomers with
S donor atom of salicylaldimine as stereo center (Fig. 3). Three peripheries of Pt in C1a and C2a
were occupied by ONS-donor atoms of salicylaldimine ligand and the fourth position was
occupied by ancillary chloride that occupied trans position to salicylaldimine N-donor atom.
-
C2b and C3b symmetry was similar to the fore-mentioned except the ancillary Cl was replaced
by 4-picoline. Selected bond lengths, angles and torsion angles of C1a, C2a, C2b and C3b were
summarized in Table 1. The sum of bond angles around Pt in all these complexes was ca. 360º
(Table 1). The bond lengths of the donor atoms of the salicylaldimine ligand and Pt center were
comparable in all these complexes (Table 1 entry 1-3). The bond length between Pt(1) and
ancillary Cl(1) in C1a and C2a were quiet matching (Table 1 entry 4) while in C2b and C3b the
bond length between Pt(1) and N(2) were equal (Table 1 entry 4). The bond angles among donor
atoms in C1a and C2a showed similarity. Similarly, C2b and C3b were similar while the bond
angles of complexes in both these group were quite different from each other. ONS-
salicylaldimine donor ligand in all these complexes occupied almost planner symmetry around
Pt(II) center.
Table 1. Selected bond length (Å), angles (°) of C1a, C2a, C2b and C3a
Entry
Bond/Angle
C1a
C2a
C2b
C3b
Bond length (Å)
1
2
3
4
Pt(1)-O(1)
Pt(1)-N(1)
Pt(1)-S(1)
Pt(1)-Cl(1)/N(2)
1.991(4)
1.995(5)
2.2136(17)
2.322(2)
1.989(5)
1.975(5)
2.212(2)
2.319(2)
1.956(6)
1.988(7)
2.226(3)
2.039(7)
1.977(3)
1.983(4)
2.2227(15)
2.039(4)
Bond angle (°)
5
6
7
∠O(1)-Pt(1)-N(1)
∠O(1)-Pt(1)-S(1)
∠N(1)-Pt(1)-S(1)
94.17(19)
176.06(15)
87.25(15)
94.9(2)
177.02(14)
87.48(16)
93.8(3)
176.44(18)
88.0(2)
94.99(14)
177.75(10)
86.92(11)

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8
9
1
∠O(1)-Pt(1)-Cl(1)/N(2)
∠N(1)-Pt(1)-Cl(1)/N(2)
∠S(1)-Pt(1)-Cl(1)/N(2)
88.56(14)
177.27(14)
90.03(7)
88.13(14)
176.86(16)
89.47(7)
85.3(3)
178.3(3)
92.9(2)
85.54(14)
175.98(15)
92.64(11)
0
Fig. 3 Single crystal plot of R and S stereo isomers observed in single crystal packing (A) C1a, (B) C2a, (C) C2b
and (D) C3b, S donor atom having a lone pair and three different atoms (Pt(1), C(9) and C(14/15)) attached showed
stereo- isomerism.
3.1.3. Stability/aquation of reference complexes C2a and C2b
Platinum complexes and approved platinum-based drugs have low or no solubility in water or
cell culture media therefore their stock solutions are prepared in dimethyl sulfoxide (DMSO) or
other organic solvent for their in vitro or in vivo biological screening [61]. Similarly platinum
anticancer complexes were exposed to aquation in solution state and in most cases the ancillary
chloride is exchanged by water or any donor co-solvent like DMSO so the solution based
investigation of their stability or aquation is important and it also affect the biological properties
of a platinum complexes [62, 63]. The ultimate target of platinum complexes is DNA or other

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biomolecules that function as a carrier for platinum complexes. The aquation of chloride
ancillary ligand prior to binding with DNA is considered an important step in interaction of a
metal complex with DNA [64, 65]. In the presence of DMSO this aquation may be replaced by
the coordination of DMSO to Pt which may alter the structure of the platinum complex and in
turn affect the anticancer activity [61].
To check this aquation and stability of the current complexes, we took C1a and C1b as reference
1
compounds and checked their stability in 17% D
2
O/DMSO-d
6
mixture using H NMR
spectroscopy. We observed different behavior in case of C1a in which we observed aquation
over the passage of time and in the aquated complex water was exchanged by DMSO (Scheme 2).
In shorter time (12 h) the equilibrium was reached and that remained constant for at least one
month (Fig. 4).
-
Scheme 2 Dissociation of C1a in DMSO/water mixture showing replacement of Cl by DMSO
-
By the replacement of Cl ligand of Pt by DMSO we observed a new set of same number of
1
peaks in slight up field or downfield for a new closely related compound. In the H NMR spectra
-
taken in pure DMSO-d
6
(Fig. 4), we did not observe such replacement of Cl even after long time
that showed water is crucial for such dissociation and showed involvement of water in this
process. To confirm this phenomenon we performed LC-HRMS analysis for similar mixture in
prepared in non-deuterated water/DMSO mixture (ESI Fig. S2-3). We found a new single charge
specie which m/z was equal to C1a-DMSO. This confirmed the aquation by water and then
replacement of water by DMSO donor ligand. Similarly we conducted HR-MS analysis for C2a

ACCEPTED MANUSCRIPT
+
in pure DMSO and we found only [M+H] peak (ESI Fig. S4). This proved in the absence of
-
water such replacement of Cl by DMSO was not possible.
2
Fig. 4 Stability analysis of C1a in 17 % D O/DMSO at mentioned time points. The new peak appearance showed
-
the displacement of the ancillary Cl ligand by DMSO or D O and the equilibrium was established at 12 h time point
2
that remained unchanged up to 30 days.
1
C2b was taken as a reference compound for “b” series of these complexes. Repeated H NMR
spectra were recorded over 30 days (ESI Fig. S1). No changes were observed in the respective
proton chemical shifts, it showed C2b was quite stable under these conditions and also denoted
that “b” series of these complexes is a highly stable group of complexes with higher anticancer
effect.
The ultimate target of Pt complexes was confirmed to be DNA and the formation of Pt-DNA
adduct, that further inhibited DNA replication and resulted in apoptosis. In some studies, it was

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shown that cisplatin is aquated inside cell or it interacted with other biomolecules like S-donor
amino acid to reach the ultimate target DNA inside nucleus and there by stop cells growth [9, 62,
6
5-67]. So considering these facts hydrolysis may be a positive property of platinum complexes
and it facilitates the interaction of these complexes (C1a-C4a) with other biomolecule and DNA.
Considering “b” series (C1b-C4b) of complexes the S-donor atom of the ligand may work as
labile group to interact with DNA. This idea may need further experimentation, which could be a
matter of further mechanistic studies.
3
.2. Results (Biology)
3.2.1. In vitro cytotoxicity analysis of C1a-C4a or C1b-C4b
To investigate the effect of C1a-C4a or C1b-C4b on cancer cell growth and proliferation, we
performed MTS assay (Fig. 5). We tested the effect of C1a-C4a or C1b-C4b in colorectal
cancer HT-29, triple negative breast cancer MDA-MB-231 and non-small cell lung cancer A549
cells. We observed that all these complexes possessed significant cytotoxic effect towards these
cancer cells in dose-dependent manner (0, 1, 3, 5 and 10 µM). The IC value for each complex
5
0
calculated in three types of cancer cells (Table 2) was far lower from that calculated for cisplatin,
showing the stronger cytotoxic property of this class of complexes as compared to cisplatin.
Fig. 5 and table 2 described cytotoxic profile of each of these complexes showing the effect of
different substituent on phenolic part of the ligand in each complex. The IC value of “a” series
5
0
of complexes (C1a-C4a) was observed in the range 2.11-2.65 µM that was about 5 times more
as compared to that of cisplatin. The anticancer effect of “b” series of complexes (C1b-C4b) was
better as compared to “a” series and cisplatin (Table 2). Among these complexes, C2b showed
the highest cytotoxicity in all kind of cancer cells. The lowest IC value (0.08 µM) for C2b was
5
0
observed in MDA-MB-231 cells.

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Furthermore, the effect of C1b, C2b and C3b was stronger in comparison to other complexes in
these cancer cells. Remarkably, cancer cells treated with C1b and C2b lead to huge cell death as
compared with other complexes or cisplatin. Low concentrations of these complexes (1 and/or 3
µM) were enough to initiate significant cell death and thereby suppress cancer cell growth.
Taking together, these results suggested that all these complexes have huge potential to suppress
cancer cell growth. Among these, the anticancer effect of C1b, C2b and C3b was higher than the
other counterparts in cancer cells.

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Fig. 5 C1a-C4a or C1b-C4b reduced cell growth ability of multiple cancer cells; MTS assay of HT-29, MDA-MB-
2
31 and A549 cells treated with various concentrations (0, 1, 3, 5 and 10 µM) of each of C1a-C4a, C1b-C4b or
cisplatin (10 µM) for 72 h and used to determine cell viability. Data from two independent experiments performed
in triplicate are shown as mean ± SD. (*P < 0.05; **P < 0.01; ***P< 0.005).
Table 2. In vitro antitumor effect(s) of C1a-C4a and C1b-C4b in various human cancer cells; IC50 (µM
Complex
C1a
C1b
C2a
C2b
C3a
C3b
C4a
C4b
cisplatin
HT-29
2.11
0.61
2.31
0.22
2.87
0.66
2.65
1.02
>10
MDA-MB-231 1.07
0.17
1.27
0.08
0.72
0.13
1.11
0.53
≥10
IC50 is defined as the concentration of a cytotoxic agent that is required for 50% inhibition of cells growth.
3.2.2. C1b-C3b induced apoptotic cell death (PARP cleavage)
Our results showed that C1b-C3b exhibited significant cytotoxic effect, therefore we decided to
carry out further experiments with these complexes. To this end, we tested the effect of these

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complexes in induction of apoptotic cell death by detection of PARP cleavage and Caspase-3/7
activation, which are significant markers of apoptosis. We treated HT-29 and A549 cells with 1
µM and 3 µM of C1b-C3b or with cisplatin (10 µM). We observed strong induction of PARP
cleavage induced by C2b and C3b in HT-29 cells (Fig. 6). In contrast, we observed activation of
PARP cleavage only by C2b in A549 cells. In both cell lines, C2b was involved in strong
activation of PARP cleavage (Fig. 6), suggesting its important role in the induction of apoptotic
cell death by PARP cleavage.
Fig. 6 Western blot analysis of HT-29 and A549 cells treated with various concentrations of C1b-C3b or cisplatin
(
10 µM) for 24 h and used to detect PARP cleavage to measure apoptotic cell death. Anti-GAPDH antibody was
used as loading and transfer control.
Next, Caspase-3/7 dependent signaling is considered important for the activation of apoptosis. In
most cases, activation of apoptotic signaling is coupled with activation of Caspase-3/7 dependent
pathway. Thus, induction of Caspase-3/7 suggested initiation of apoptosis. Therefore, we
decided to check the effect of these complexes on the induction of Caspase-3/7 dependent
signaling in cancer cells. Importantly, we observed strong activation of Caspase-3/7 in response
to C1b-C4b in both HT-29 and A549 cells (Fig. 7). These results points towards the role of these
complexes in the initiation of apoptosis by Caspase-3/7 dependent pathways, suggesting that
C1b-C4b have the ability to activate apoptotic signaling pathways through Caspase-3/7
activation. Taking together, these results from Fig. 6 and Fig. 7 showed that these complexes
induce apoptotic cell death by activation of PARP cleavage and Caspase-3/7.

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Fig. 7 Caspase-3/7 activity assay; HT-29 and A549 cells were treated with various concentrations of C1b-C4b or
cisplatin (10 µM) for 24 h and used to determine the apoptotic cell death. Experiments were repeated three times in
duplicate and similar results were obtained. Data are shown as mean ± SD (*P < 0.05; **P < 0.01; ***P< 0.005).
3.2.3. C1b-C4b suppressed colony formation ability of cancer cell
Cancer cells have the ability to form colonies due to uncontrolled cell growth coupled with
abnormal DNA synthesis. Colony formation ability of cancer cells often leads to tumor burden in
vivo and in most cases these colonies show resistance to cancer drugs. Activation of survival
signaling plays key role in the clonogenic formation ability of cancer cells. Therefore, we tested
the effect of C1b-C4b on the clonogenic formation ability of HT-29 and A549 cells.
Interestingly, we observed that different doses of C1b-C4b were able to inhibit the colony
formation ability of both HT-29 and A549 cells in comparison to cisplatin (Fig. 8). These
complexes seemed to exert strong cytotoxic effect and thereby suppress their clonogenic

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formation ability in these cells, suggesting their potentials to block the colony formation ability
of cancer cell and play important role in the induction of apoptotic.
Fig. 8 Colony formation assay; A549 and HT-29 cells were treated with various concentrations of C1b-C4b or
cisplatin (0.25 µM) for 24 h and used to determine the long-term response. Representative photograph of clonogenic
assay results are shown. Experiments were repeated three times and similar results were obtained.
3.2.4. C1b-C3b regulate MAPK signaling and p21 expression
Next, we determined the effect of C1b-C3b on the regualtion of mitogen activated protein
kinases (MAPK) and p21 expression. To this end, we selected ERK1/2 and AKT which are parts
of MAPK signaling. Intersetingly, we found that the effect of these complexes on the regulation
of both ERK1/2 and AKT signaling was not obviuos (Fig. 9A), representing that these
complexes may not target these signaling pathhways and may exert effect in context-dependent
manner (Fig. 9A). Furthermore, we tested the effect on p21 expression in cancer cells treated
with C1b-C3b. p21 is a tumor suppressor protein, which plays critical role in the inhibition of
cell cycle progression by blocking DNA synthesis. We observed strong induction of p21 protein
levels in both HT-29 and A549 cells, when traeted with these complexes (Fig. 9B). p21
activation suggested that C1b-C3b have potential to suppress cell cycle progression by inhibiting