Investigation of triazole-linked indole and oxindole glycoconjugates as potential anticancer agents: Novel Akt/PKB signaling pathway inhibitors

Article abstract of DOI:10.1039/c5md00513b

In continuation of our venture towards the synthesis of novel bioactive agents, two sets of triazole-linked glycoconjugates were synthesized from indole/oxindole (29 compounds) and were further characterized by IR (infrared spectroscopy), ¹H NM

Full text of DOI:10.1039/c5md00513b

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PAPER
Investigation of triazole linked indole and oxindole
glycoconjugates as potential anticancer agents: Novel Akt/PKB
signaling pathway inhibitors†‡
Received 00th January 20xx,
Accepted 00th January 20xx
Atulya Nagarsenkarɠ,a, Santosh Kumar Prajaptiɠ,a, Sravanthi Devi Guggilapua, Swetha Birinenib,
Sudha Sravanti Kotapallib, Ramesh Ummannib*and Bathini Nagendra Babua*
DOI: 10.1039/x0xx00000x
www.rsc.org/
In continuation of our venture towards the synthesis of novel bioactive agents, the two sets of triazole linked
glycoconjugates were synthesized respectively from indole/oxindole (29 compounds) and were further characterized by IR
(infrared spectroscopy), 1H NMR (nuclear magnetic resonance), 13C NMR and mass spectral analysis. The newly synthesized
target compounds were evaluated for their preliminary in vitro anticancer activity against DU145 (prostate cancer), HeLa
(cervical cancer), A549 (lung cancer) and MCF-7 (breast cancer) cell lines. In the sulforhodamine B (SRB) assay, the results
indicated that the compounds 5f (indole derivative) and E-9b (oxindole derivative) displayed remarkable cytotoxic activity
against the DU145 cells. Moreover, the colony formation assay (soft agar assay) revealed that the compounds 5f and E-9b
can inhibit the growth and proliferation of DU145 cells. The impact of the most active cytotoxic compounds 5f and E-9b on
the cell cycle distribution was assessed in DU145 cells, which displayed a cell cycle arrest at the sub-G1 phase. Next, the
compounds 5f and E-9b were tested for caspase activation in the DU145 cells, and the results specified that the these
compounds have capability to induce apoptosis in cells through an intrinsic pathway leading to subsequent cell death.
Further studies also confirmed that the compounds 5f and E-9b act against protein kinase B (Akt/PKB) pathway to inhibit
proliferation of cancer cells. Thus, the compounds 5f and E-9b could be the novel potential anticancer leads as the normal
cell line NIH/3T3 (fibroblast) studies showed no significant cytotoxicity.
growth inhibition of tumor cells with an increased amount of Akt.
The observed reliance of certain tumors on Akt signaling for survival
and growth has broad implications for cancer therapy, offering the
Introduction
Nowadays, cancer has gradually become the leading cause of
mortality worldwide, seriously endangering the health and life of
humans for a long period. It has accounted for 8.5 million deaths
(around 17% of all deaths) in 2012, and estimated to cause 14
million deaths in 2030.1 Despite the huge efforts to implement
novel chemotherapeutic strategies for the successful treatment of
different types of cancer, this disease still remains as one of the
major concerns globally.2 Hence, there is an urgent need to search
for some newer and safer anticancer agents that have broader
spectrum of cytotoxicity towards tumor cells.
prospective for targeted tumor cell killing.5 In the last few years,
through combinatorial chemistry, high-throughput, virtual
screening, and traditional medicinal chemistry,
inhibitors of the Akt pathway have been identified.6
a number of
The 3-substituted indole and oxindole scaffolds possess various
biological activities and continues to be a main structural unit of
many natural and pharmacologically active compounds (Figure 1).7-9
Recently, new compounds bearing 3-substituted indole and
oxindole moieties designed to target Akt pathway have been
described.10,11
The protein kinase B (Akt/PKB) pathway is an important new target
for molecular therapeutics, as it functions as a key nodal point for
transducing extracellular (growth factor and insulin) and
intracellular (receptor tyrosine kinases, Ras and Src) oncogenic
signals.3,4 Obstruction of Akt signaling results in apoptosis and
Molecular hybridization which covalently combines two or more
drug pharmacophores into a single molecule is an effective tool to
design more active novel chemical entities.12-14 Besides, the hybrids
may also minimize the redundant side effects and allow synergic
action.15,16 On the other hand, the copper-catalyzed azide–alkyne
cycloaddition (CuAAC) with carbohydrate based azide substrates is
now usually applied in drug discovery and has proven exceptionally
pliable both in the preparation of glycoconjugate small molecule
inhibitors against a range of enzymes and to affix carbohydrates to
biomolecules.17 Glycopyranosyl have advantages of being stable
and are inert towards a wide range of reaction conditions and most
importantly, they are readily synthesized anomerically pure.18
ɠAuthors contributed equally to this work.
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical
Education and Research, Balanagar, Hyderabad, 500037, India. *E-mail: bathini.
niperhyd@gov.in; Tel.: +91-40-23073740; fax: +91-40-23073751
bCenter for Chemical Biology, Indian Institute of Chemical Technology (CSIR-IICT),
Tarnaka, Hyderabad 500007, India
† The authors declare no competing interests.
‡ Electronic Supplementary Information (ESI) available: See
DOI: 10.1039/x0xx00000x
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In this context, we desire to exploit azidosugars as the diversity oxindoles were next subjected to N-propargylation for the
surrogates to couple with the alkyne-modified 3-substituted synthesis of respective alkynes E-8(a-j) and Z-8(a-j). These
indoles and oxindoles. These 3-substituted indole and oxindole terminal alkynes bearing substrates E-8(a-j) and Z-8(a-j) were
derivatives may have the advantages of structural diversity, further subjected to [3+2] cycloaddition with β-D-
improved solubility and most important tunable anticancer glucopyranosyl azide (
2
) to afford triazole linked oxindole
activity.
glycoconjugates E-9(a-j) and Z-9(a-j)
.
Herein, the triazole linker serves as a biocompatible, non-labile
covalent spacer between the acetylated glucose and the
substituted indole/oxindole.
HO
O
N
N
OAc
OAc
F
N
N
O
O
H
AcO
AcO
H
AcO
AcO
OAc
N3
HN
OAc
OAc
O
O
N
H
1
2
N
H
R1
N
H
(c)
O
(b) Semaxanib
R
(a)
Sunitinib
O
R
HN
R1
Dacinostat
OAc
R1
i
ii
OH
O
N
N
R
O
N
N
N
N
N
AcO
O
N
N
AcO
N
H
H
N
N
OAc
3 (a-k)
4 (a-k)
n(HO)
5 (a-k)
N
OH
OH
O
O
N
N
O
O
SMe
Cl
Indibulin (D-24851)
F
N
R =
(j)
R =
R
(f)
(h)
(i)
R =
OH
N
O
(a) R = -CHO, R1 = H
Carbohydrate cyclopamine
glycoconjugate
N
(d)
(e)
(f)
R1 = H
R1 = H
R1 = H
H
(b) R = -CHO, R1 = -OCH3
(c) R = -CH=N-OH, R1 = H
(d) R = -COCH3, R1 = H
(e) R = -NO2, R1 = H
O
(g)
R =
F
Fig. 1:
Structure of some representative bioactive compounds containing indole, oxindole,
triazole and glycosyl triazole moieties
R =
R1 = H
(k)
R =
O
O
R1 = H
N
H
N
H
R1 = -OCH3
Scheme 1:
Synthesis of 3-substituted indole triazole linkedglycoconjugates;Reagents
and conditions:(i) propargylbromide,K2CO3, acetonitrile,reflux, 6 h; (ii)D-
glucopyranosylazide,CuI, t-butanol/water(1:1), 40 oC, 48 h.
Results and discussion
Chemistry
R
R
The synthesized glycoconjugate compounds of the present
study comprises three core structural elements: (i) a sugar
moiety (β-D-glucopyranoside), (ii) a 1,2,3-triazole ring, and (iii)
a substituted indole/oxindole. The target glycoconjugates 5(a-
OAc
iii
iv
R
O
N
N
N
O
O
AcO
AcO
N
N
H
N
OAc
O
E-7 (a-j)
E-8 (a-j)
E-9 (a-j)
ii
R
i
k), E-9(a-j) and Z-9(a-j) were synthesized in two sets of
O
O
N
H
N
H
7 (a-j)
molecules using substituted indole and oxindole as starting
materials.
6
ii
R
OAc
R
In the first series (Scheme 1), substituted indoles 3(a-k) were
subjected to N-propargylation in the presence of a base to give
respective alkynes 4(a-k). These alkynes were further reacted
O
iv
N
N
iii
AcO
AcO
O
O
N
N
R
N
N
H
OAc
O
Z-9 (a-j)
Z-8 (a-j)
Z-7 (a-j)
with β-D-glucopyranosyl azide (
Huisgen 1,3-dipolar cycloaddition to furnish triazole linked
indole glycoconjugates 5(a-k)
2) via copper(I)- catalyzed
R=
(a)
(g)
(h)
(d)
(e)
N
S
.
(b)
N
For the preparation of azide precursor, required for the
synthesis of proposed glycoconjugates, we have utilized
O
(i)
(j)
MeO
(c)
(f)
MeO
commercially
available
1,2,3,4,6-penta-O-acetyl-D-
Scheme 2: Synthesis of 3-substituted oxindole triazole linked glycoconjugates; Reagents and
conditions: (i) aldehyde, piperidine, ethanol, reflux, 3-4 h; (ii) isomers separated
by using column chromatography; (iii) propargyl bromide, K2CO3, acetonitrile,
reflux, 6 h; (iv) D-glucopyranosyl azide, CuI, t-butanol/water (1:1), 40 oC, 48 h.
glucopyranose (1) as the starting material. The β-D-
glucopyranosyl azide (2) was synthesized via bimolecular
displacement of the halide substituent of glycosyl halide
precursor with an azide nucleophile (Scheme 1).19
Biological evaluation
In the second series (Scheme 2), the well-known Knoevenagel
condensation was employed on oxindole by using different
aromatic, heteroaromatic and aliphatic aldehydes to afford 3-
alkenyl oxindoles 7(a-j) in a mixture of E/Z isomeric forms.20,21
These E/Z isomers were separated by using the conventional
column chromatography and were characterized with the help
of 1H NMR. The chemical shift values of the separated isomers
[E-7(a-j) and Z-7(a-j)] were carefully compared with previous
literature.22-28 The compound E-7g was obtained in selective E
form as reported in the literature,26 whereas the compound Z-
7c was formed as the Z isomer with negligible amount of the E
isomer.24 In the next step, these isomerically pure 3-alkenyl
In vitro anticancer activity. All the newly synthesized compounds
were evaluated for their in vitro cytotoxic activity against DU145,
HeLa, A549 and MCF-7 cancer cell lines by employing the
sulforhodamine B (SRB) assay.29 Doxorubicin was used as the
reference for this study. The concentration response course analysis
was performed to determine drug concentrations required to
inhibit the growth of cancer cells by 50% (IC50) after incubation for
48 h. The results from the in vitro anticancer studies revealed that
the synthesized target compounds exhibited different levels of
anticancer properties (Table 1, Table 2). From the close examination
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of the IC50 values (Table 1), it was observed that the indole showed inhibition against all tested cancer cells. Both E- and Z-
glycoconjugate substituted with –CHO functional moiety at position isomers of oxindole glycoconjugate with the N,N dimethyl phenyl
3 (5a) showed inhibitory activity against DU145, HeLa and MCF-7, substitution (E-9e and Z-9e) showed excellent activity towards all
while no activity was observed in the case of A549 cells. The cancer cell lines. The E isomer of naphthalenyl substituted analogue
compound substituted with -CHO and -OMe at positions 3 and 5 of (E-9f) showed moderate activity against DU145, A549, and MCF-7,
indole respectively showed decrease in the activity for DU145 and whereas its Z-form (Z-9f) lost its activity against DU145 and HeLa
A549 cell lines, whereas complete loss of inhibition was seen cell lines. The E-isomer of pyridinyl substituted compound (E-9g)
against HeLa and MCF-7 cell lines (5b). The glycoconjugate displayed modest anticancer activity. The thiophene substituted E-
substituted with oxime (5c) showed inhibitory activity especially isomer of oxindole glycoconjugate (E-9h) showed moderate to good
against DU145 and MCF-7 cells. On the other hand, the indole inhibitory activity against all cancer cell lines. However, complete
glycoconjugate substituted with the acetyl group (5d) displayed loss of activity was seen against the HeLa in case of Z-isomer (Z-9h).
modest activity specifically against DU145 cell line. The compound Furthermore, the E-isomer of furyl substituted analogue (E-9i)
with nitro substitution at position 3 of the indole displayed exhibited good inhibition against all cancer cell lines, whereas
moderate activity towards DU145, A459, and MCF-7 cell lines (5e). activity of its Z-isomer was found to be insignificant (Z-9i). In case of
Interestingly, the cross aldol product substituted with the phenyl the oxindole glycoconjugates substituted with aliphatic chain, the E-
ring (5f) showed enhancement in the anticancer activity towards all isomer (E-9j) showed promising results while its Z-isomer (Z-9j) led
tested cancer cell lines. However, the bulky group substituted cross to decrease in the anticancer activity. All compounds were tested
aldol product of glycoconjugate (5g) led to the decreased against
a non-cancerous cell line NIH/3T3 (fibroblast). The
anticancer activity, while the pyridine substituted analogue (5h) compounds Z-9b and Z-9i were found to be cytotoxic against
demonstrated improvement in the activity for DU145, A549 and NIH/3T3 cells, while the compounds 5f and E-9b displayed
MCF-7 cell lines. Using oxindole, an attempt was made to promising results in case of non-cancerous cell as compared to their
synthesize the Knoevenagel product (5i) of the glycoconjugate 5a, reference drug. The compound 5f was found to be inactive against
which displayed inhibition specifically against the MCF-7 cell line. NIH/3T3 cells, whereas the compound E-9b showed IC50 against the
While, the glycoconjugate substituted with 5-fluoro oxindole at same cell line at 73.3 µM.
position 3 of indole (5j) showed modest inhibition towards DU145
Table 2. In vitro anticancer activity of triazole linked oxindole
glycoconjugates derivatives (IC50 µM)
and A549 cell lines. The indole compound (5k) substituted with 5-
fluoro oxindole at the 3rd and methoxy at the 5th position was found
C
DU145
A549
HeLa
MCF-7
NIH/3T3
to be inactive towards all tested cancer cell lines.
E-9a
22.8±10.1
20.6±5.3
37.1±3.5
30.2±1.7
NA
Z-9a
E-9b
24.1±3.6
9.3±0.5
17.9±2.5
16.0±0.4
33.3±4.8
85.6±8.5
24.3±5.2
NA
22.8±2.0
73.3±3.6
Table 1. In vitro anticancer activity of triazole linked indole
glycoconjugates derivatives (IC50 µM)
Z-9b
Z-9c
E-9d
Z-9d
9.4±0.8
24.9±7.2
17.1±2.0
19.5±3.9
16.5±1.6
23.3±0.1
NA
NA
NA
9.0±1.2
NA
C
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
3f
1
DX
DU145
58.7± 8.2
136.1±5.3
88.4±10.2
75.2±13.6
60.9±9.4
14.4±0.5
58.1±12.6
29±2.8
NA
87.4±5.3
NA
NA
NA
5.4±0.03
A549
NA
106.3±8.3
NA
HeLa
44.5±4.3
NA
NA
NA
MCF-7
25.2±2.1
NA
51±3.7
NA
NIH/3T3
NA
NA
26.2±0.2
NA
20.5±2.1
NA
NA
NA
NA
62.5±2.6
NA
63.4±0.5
NA
23.5±0.6
53.2±8.8
34.1±5.8
12.3±5.2
25.9±6.3
58.4±29.3
98.1±5.6
NA
E-9e
Z-9e
11.2±0.9
19.4±3.5
17.7±2.0
26.3±3.8
25±5.4
25.2±2.2
29.8±2.4
12.7±3.2
17±0.09
93.2±6.7
16.3±2.3
46.8±5.0
39.8±1.6
NA
36.9±6.5
NA
NA
NA
NA
10.1±0.03
33.0±4.8
165.2±7.9
NA
NA
NA
NA
NA
NA
7.2±0.1
23.9±3.5
44.0±0.3
22.3±2.3
22.2±1.9
40.4±1.9
NA
E-9f
Z-9f
E-9g
E-9h
Z-9h
43.7±2.3
NA
48.3±2.0
23.4±0.7
22.8±0.2
30.9±6.3
62.2±7.1
26.1±2.8
20.8±1.4
16.1±1.9
NA
NA
51.7±0.6
36.9±2.2
NA
32.7±1.6
46.5±6.7
32.1±7.5
28.1±1.8
36.1±10.8
28.4±1.2
80±6.9
NA
48.1±3.5
14.6±2.5
NA
NA
NA
NA
E-9i
Z-9i
10±0.3
14.5±0.05
41.1±10.8
29.9±2.1
NA
29.1±2.4
38.2±9.4
31.8±1.6
13.3±1.4
NA
7.1±0.01
16.9±1.1
6.1±0.9 10.1±0.03
E-9j
Z-9j
E-7b
DX
19.7±3.9
26.6±5.3
NA
24.1±1.8
40.4±13.1
NA
NA
46.3±4.3
NA
22.6±5.8
25±2.4
NA
NA
NA
NA
C: Compound; NA: Not active (IC5o > 200 µM); DX: Doxorubicin
In the second set of the glycoconjugates (Table 2), it was observed
that the phenyl substituted E- and Z- isomers of oxindole
glycoconjugates (E-9a and Z-9a) showed almost similar activities
against all tested cancer cell lines. Enhancement in the inhibitory
activity was observed in case of tolyl substituted E- and Z- isomers
(E-9b and Z-9b) selectively towards DU145 cell line, whereas
complete loss of activity was observed against the MCF-7 cells. The
compound substituted with 3,4-dimethoxy phenyl moiety (Z-9c)
displayed good inhibition against all tested cancer cell lines. E-
isomer of cinnamyl substituted analogue (E-9d) was found to be
active only against DU145 and MCF-7, while its Z-isomer (Z-9d)
5.4±0.03
7.1±0.01
7.2±0.1
6.1±0.9
10.1±0.03
C: Compound; NA: Not active (IC5o > 100 µM); DX: Doxorubicin
Additionally, the building blocks of target compounds i.e. acetylated
glucose (1), 3-substituted oxindole (E-7b) and 3-substituted indole
(3f), were screened for their anticancer potential and were found to
be inactive against all tested cell lines. These results suggest that
the enhancement of anticancer activity occurs in case of triazole
linked indole and oxindole glycoconjugates rather than the
individual units of the target compounds.
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Analysis of the SRB assay results suggests DU145 cells are more Colony Formation (Soft Agar) Assay. Prostate cancer cells have an
sensitive towards both the series of compounds. The impact of ability of continuous proliferation. To test whether the compounds
modification of the “R” group in indole and oxindole derivatives is affect this property of the cells, a colony formation assay (soft agar
interesting in the light of the results of the SAR study, which assay) which mimics an in vivo environment was conducted.30 From
suggests that substituted phenyl moiety at 3rd position is optimal the assay, it was clearly demonstrated that the compounds 5f and
for anticancer activity. On the other hand indole and oxindole E-9b inhibit the growth of cells in a dose dependent manner.
derivatives bearing bulky substitutions at this position It was clearly observed that the compounds inhibit anchorage-
demonstrated decrease in the activity. Further derivatization mostly independent growth of the cells, when the cells were treated with
resulted in a significant loss of activity. Among all compounds the compounds in increasing concentrations. Thus, the long term
synthesized, compounds 5f and E-9b showed the most significant effects of the compounds 5f and E-9b were confirmed on DU145
cytotoxicity to cancer cell lines and were comparatively safer (prostate cancer cells). The inhibition of growth and proliferation
against NIH/3T3 cells. These preliminary results encourage further
investigation on the synthesized compounds aiming to the
development of novel potential anticancer agents.
Change in morphology. Out of all the compounds in the series
synthesized, compounds 5f and E-9b were found to have promising
anti-cancer activity, especially against DU145 (prostate cancer) cell
line. As these compounds were cytotoxic towards DU145 cells with
IC50 values of 9.3 µM (E-9b) and 14.4 µM (5f), we further tested
their ability to affect the morphology of the cells.30 The cells were
was observed at 5 μM in case of cells treated with compound 5f
(Figure 3A), while the inhibition was seen at 10 μM and higher
concentrations in cells treated with compound E-9b (Figure 3B).
treated with the compounds 5f and E-9b respectively in increasing
concentrations. After 48 h of incubation, it was observed that the
cells began to lose their characteristic morphology (Figure 2A and
2B). Thus, it was confirmed that the compounds have an effect on
the morphology of cells.
Fig. 3A: Effect of 5f on the inhibition of colony formation and growth of cells when the
cells were treated with 5f. DMSO was taken as a control. Representative images of the
colony-forming assay are shown here. The ability of the cells to proliferate and form
colonies decreases with increasing concentration of 5f.
Fig. 2A: DU145 cells were treated with 5f at the given concentrations and DMSO taken
as a control. After 48 h of incubation, the morphology of the cells changes with
increasing concentration of compound.
Fig. 3B: Long term effect of E-9b on the colony-forming ability of DU145 cells. DU145
cells were treated with indicated concentration of E-9b and were allowed to grow for 9
days to form colonies. Representative images of the colony-forming assay are shown
here. Number of colonies and their size formed by DU145 in soft agar is decreased on
exposure to E-9b. DMSO was taken as a control.
Cell cycle analysis. Many of the cytotoxic compounds exert their
growth inhibitory effect by arresting the cell cycle at a specific
checkpoint.31 In vitro screening results revealed that the
compounds 5f and E-9b displayed significant activity against DU145
cells.
Fig. 2B: DU145 cells were treated with E-9b at the given concentrations and DMSO
taken as a control. After 48 h of incubation, the morphology of the cells changes with
increasing concentration of compound.
4 | J. Name., 2012, 00, 1-3
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control. It can be observed from the images that the cells accumulate in sub-G1 phase
with increasing concentrations as compared to DMSO.
Table 4: DU145 cells were treated with E-9b at varying
concentrations (5 µM, 10 µM, 15 µM, 20 µM and 25 µM). The cells
were harvested and analyzed via flow cytometry. Data represents the
cell population (%) in each cell cycle phase.
E-9b
Sub G1 (%)
G0/G1 (%)
S (%)
G2/M (%)
DMSO
5 µM
10 µM
15 µM
20 µM
25 µM
2.30
7.12
14.40
17.20
34.34
37.51
61.28
59.15
48.07
46.65
40.46
41.35
8.62
6.85
7.56
7.64
5.72
4.86
15.81
12.62
16.73
14.89
10.89
9.05
Determination of activation of caspase-3, caspase-9 and caspase-
8. Cell cycle arrest and inhibition of cell growth mostly leads to the
cellular apoptosis. Therefore, in order to investigate whether the
compounds induce apoptosis in DU145 cells, caspase activation in
cells was studied. Caspases are crucial mediators of programmed
cell death (apoptosis). Among them, the caspase-3 is a frequently
activated death protease; catalyzing the specific cleavage of many
key cellular proteins during apoptosis.32 It has been previously
proven that the activation of caspase-3 occurs in a sequential
manner. Caspase-8 and caspase-9 are known to be involved in the
activation of caspase-3. Both Caspase-8 and caspase-9 are
considered as the initiator caspases.33,34 To measure the activation
of caspases, fluorescence substrates were used. Ac-DEVD AMC was
used as the substrate for caspase-3, while Ac-DEVD AFC and Ac-
VETD-AMC were used to determine activation of caspase-9 and
caspase-8 respectively.
Fig. 4A: Cell cycle analysis of cells treated with 5f. The cells were stained with
propidium iodide and subjected to flow cytometry (FACS). DMSO was taken as a
control. It can be observed from the images that the cells accumulate in sub-G1 phase
with increasing concentrations as compared to DMSO, with significant increase at 10
µM concentration.
Table 3: DU145 cells were treated with 5f at varying concentrations (5
µM, 10 µM, 15 µM, 20 µM and 25 µM). The cells were harvested and
analyzed via flow cytometry. Data represents the cell population (%) in
each cell cycle phase.
5f
Sub G1 (%)
2.30
G0/G1 (%)
61.28
75.04
21.19
21.46
S (%)
8.62
4.72
4.41
3.82
1.34
0.80
G2/M (%)
15.81
8.22
16.16
11.91
1.87
DMSO
5 µM
10 µM
15 µM
20 µM
25 µM
4.53
43.68
54.02
82.38
84.06
12.91
11.96
1.01
Therefore, we herein examined the effect of the compounds 5f and
E-9b on DU145 cell cycle using flow cytometric analysis. DU145 cells
were treated with various concentrations of compounds 5f and E-
9b, stained with propidium iodide.
From the assay it was observed that the caspase-8 (Figure 5)
and caspase-9 (Figure 6) in cells were activated in response to
the treatment with compounds 5f and E-9b in a dose
dependent manner. As expected, the caspase-3 was also seen
to be activated in cells treated with compounds 5f and E-9b
(Figure 7). The activation of caspase-9 is implicated in the
induction of apoptosis via mitochondrial mediated intrinsic
pathway. Thus, it was evidently confirmed that the compounds
5f and E-9b have the ability to induce apoptosis in cells via an
intrinsic pathway leading to activation of caspase-3 and
eventual cell death.
Flow cytometry (FACS) was conducted to analyze the cells. From the
analysis (Table 3), it was observed that the cells were accumulated
in the sub-G1 phase of the cell cycle, with significant increase in cell
population at 10 μM concentration in the case of 5f (Figure 4A).
From the analysis of cells treated with compound E-9b (Table 4), it
was observed that the cells accumulated in sub-G1 phase in a
significant manner at 20 μM concentration (Figure 4B). Thus, it was
clearly demonstrated that the compounds 5f and E-9b cause cell
cycle arrest, consequently inhibiting growth and eventual
proliferation of cells
Fig. 5: Activation of caspase-8 was also observed in cells treated with compounds
E-9b and 5f. After incubation period of 48 h, it was seen that both the
compounds cause activation of caspase-8. Doxorubicin (10 µM) was used as a
control for activation of caspases. All experiments were carried out in triplicates
and mean values are presented here.
Fig. 4B Cell cycle analysis of cells treated with E-9b. The cells were stained with
propidium iodide and subjected to flow cytometry (FACS). DMSO was taken as a
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bind and inhibit the cyclin E- or cyclin A-associated cyclin-
dependent kinases (CDKs) 2 and other CDKs, and negatively
regulate G1–G2 cell cycle progression.34 From the analysis, it was
observed that the expression of p27 increased with increasing
concentrations of the compounds 5f and E-9b (Figure 8). This
markedly demonstrates that the compounds 5f and E-9b act against
the cell cycle progression through stabilization of p27Kip1 in the
cancer cells. Thus, it was further confirmed that the compounds
induce cell cycle arrest and cause eventual apoptosis. We have also
analyzed the relative expression of phosphorylated Akt (pAkt) and
p27. The expression of pAkt was compared with expression of Akt,
whereas p27 Kip1 expression was compared against constitutive β-
Actin expression (Figure 9).
Fig. 6: Relative activation of caspase-9 in cells treated with compounds E-9b and 5f
respectively. Upon 48 h of incubation it was seen that activation of caspase-9 occurs
with increasing concentrations of the compounds. Doxorubicin (10 µM) was used as a
control for activation of caspases. All experiments were carried out in triplicates and
mean values are presented here.
Fig. 8: Upon treatment of cells with 5f and E-9b, the protein expression levels of pAkt
decreased as compared to Akt. The expression of p27Kip1 protein increased with
increasing concentrations of compounds 5f and E-9b respectively. β-Actin was
considered as a control.
Fig. 7: DU145 cells were treated with increasing concentrations of E-9b and 5f
respectively. Upon 48 h of incubation it was seen that activation of caspase-3 occurs
with increasing concentrations of the compounds. Doxorubicin (10 µM) was used as a
control for activation of caspases. All experiments were carried out in triplicates and
mean values are presented here.
Akt/PKB and p27 protein expression analysis via western blot. As
discussed earlier, the compounds 5f and E-9b were found to have
an anti-proliferative effect on DU145 cells. The colony formation
assay suggests that the compounds have the ability to inhibit
anchorage-independent cell growth. Primarily, anchorage-
independent cell growth occurs by the activation of Akt/PKB
pathway.35-38 This prompted us to conduct a study to determine the
effects of compounds 5f and E-9b on activation and inactivation of
proteins involved in cancer cell proliferation. Thus, the effects of
the compounds on the activation of Akt by phosphorylation were
measured by using western blotting technique. From the study, a
significant decrease in the phosphorylation of Akt in DU145 cells
was observed upon the exposure of compounds 5f and E-9b (Figure
8). These results clearly suggest that the Akt pathway could be the
probable target for inhibition of growth in cancer cell. It has been
previously reported that the increased phosphorylation of Akt
(pAkt) in tumor samples from patients has been associated with
disease progression and increased proliferation.32 However,
compounds 5f and E-9b might be targeting Akt directly or other
Fig. 9: Relative quantitative analysis of p27 Kip 1 protein expression as compared to
constitutive expression of β-Actin protein and pAkt expression in comparison to the Akt
expression for cells treated with 5f and E-9b respectively. Expression of proteins in cells
treated with DMSO was taken as control.
Conclusion
In the current study, two different sets of triazole linked indole and
oxindole glycoconjugates were synthesized, and were further
evaluated for their in vitro anticancer potentials. An initial screening
was performed against DU145, HeLa, A549 and MCF-7 cancer cell
lines. In preliminary SRB assay, the compound 5f from indole series
and the compound E-9b from oxindole series were found to be
most active against the DU145 (prostate cancer) cell line.
Remarkably, the compounds 5f and E-9b were found to be non-
cytotoxic towards normal cell line NIH/3T3. Next, it was seen that
the compounds 5f and E-9b have an effect on the morphology of
signalling pathways indirectly. Therefore,
a precise molecular
mechanism for the inhibition of pro-proliferative pathway by the
compounds 5f and E-9b still remains to be determined.
From the earlier results, it was confirmed that the compounds 5f
and E-9b have the ability to affect the cell cycle progression in
DU145 cells. From the flow cytometric analysis, it was found that
both the compounds 5f and E-9b cause cell cycle arrest at sub-G1
phase of the cell cycle. Thus, to further verify, we have measured
the expression of cell cycle inhibitor p27Kip1 (p27). p27 is known to
6 | J. Name., 2012, 00, 1-3
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ARTICLE
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DOI: 10.1039/C5MD00513B
TABLE OF CONTENTS
TEXT
Novel triazole linked indole and oxindole glycoconjugates as inhibitors of
Akt/PKB signaling pathway
GRAPHIC