Inhibition of Invasion in Pancreatic Cancer Cells by Conjugate of EPA with β3,3-Pip-OH via PI3K/Akt/NF-kB Pathway

Article abstract of DOI:10.1021/acsmedchemlett.5b00257

The present work describes the anti-invasive effect of conjugate BC06, a novel conjugate of EPA, (2E,4E)-4-(benzo[d][1,3]dioxol-5-ylmethylene) hex-2-enoic acid with β,β-disubstituted-β-amino acid, β^3,3-Pip-OH (2-(4-aminopiperidin-4-yl)acetic acid), in human pancreatic carcinoma. The conjugate BC06 inhibited invasion and migration of PANC-1 cells in wound healing, matrigel invasion, and gelatin degradation assays. Apart from suppressing PI3K/Akt/NF-kB signaling, which is involved in the up-regulation of matrix metalloproteinases, our study also demonstrated that dose-dependent treatment of BC06 results in the upregulation of TIMP-1 and E-cadherin expression. Further, BC06 was found to be inhibiting the metastatic ability of PANC-1 cells by reducing MMP-2 and MMP-9 expression. These findings suggest that EPA conjugate with β^3,3-Pip-OH, BC06, may be used as an anti-invasive agent against human pancreatic carcinoma.

Full text of DOI:10.1021/acsmedchemlett.5b00257

Letter
pubs.acs.org/acsmedchemlett
Inhibition of Invasion in Pancreatic Cancer Cells by Conjugate of EPA
3,3
with β -Pip-OH via PI3K/Akt/NF-kB Pathway
†
,∥
‡,∥
§,∥
†
,†
†
Hina Amin, Naiem Ahmad Wani, Saleem Farooq, Debasis Nayak, Souneek Chakraborty,
‡
†
§
,‡
Sudha Shankar, Reyaz ur Rasool, Surinder Koul, Anindya Goswami,* and Rajkishor Rai*
†
‡
§
Cancer Pharmacology Division, Medicinal Chemistry Division, and Bioorganic Chemistry Division, CSIR-Indian Institute of
Integrative Medicine, Canal Road, Jammu Tawi, J&K 180001, India
*
S Supporting Information
ABSTRACT: The present work describes the anti-invasive
effect of conjugate BC06, a novel conjugate of EPA, (2E,4E)-4-
(
benzo[d][1,3]dioxol-5-ylmethylene) hex-2-enoic acid with
3
,3
β,β-disubstituted-β-amino acid, β -Pip-OH (2-(4-aminopiper-
idin-4-yl)acetic acid), in human pancreatic carcinoma. The
conjugate BC06 inhibited invasion and migration of PANC-1
cells in wound healing, matrigel invasion, and gelatin
degradation assays. Apart from suppressing PI3K/Akt/NF-kB signaling, which is involved in the up-regulation of matrix
metalloproteinases, our study also demonstrated that dose-dependent treatment of BC06 results in the upregulation of TIMP-1
and E-cadherin expression. Further, BC06 was found to be inhibiting the metastatic ability of PANC-1 cells by reducing MMP-2
3,3
and MMP-9 expression. These findings suggest that EPA conjugate with β -Pip-OH, BC06, may be used as an anti-invasive
agent against human pancreatic carcinoma.
KEYWORDS: β,β-disubstituted-β-amino acid, piperic acid, metastasis, AKT, invasion
ancreatic cancer remains the fourth leading cause of all
P
cancer deaths in most developed countries including
1
,2
United States. Phosphatidylinositol 3-kinase (PI3K)/AKT
pathway is constitutively activated in most pancreatic cancers
3
−5
and offers effective targets for therapeutic development.
Akt
is involved in the progression of metastasis through a wide
5
variety of signaling molecules and mechanisms. Further,
transcription factor nuclear factor-kB (NF-kB), which is a
direct target of Akt, has been correlated with increased
metastatic potential in pancreatic cancer and has been
6
,7
associated with metastasis and invasion in many tumors.
Recent literatures demonstrate that NF-kB is constitutively
8
activated in most pancreatic cancer cells, in animal models for
9
pancreatic cancer, and in human pancreatic cancer tissues as
8
well. NF-kB promotes the migration and invasion of pancreatic
L
8
Figure 1. Chemical structure of conjugates of EPA with Ala (BC01),
cancer cells by a number of NF-kB-regulated gene products.
L
L
3,3
3,3
Phe (BC02), Pro (BC03), β -Ac c (BC04), Gpn (BC05), and β ,
6
Unfortunately, the tolerable and specific inhibitors of the PI3K/
Akt pathway are lacking until date to target pancreatic cancer.
The present study unveils the identification of novel inhibitors
of the PI3K/Akt pathway based on conjugation of EPA,
2E,4E)-4-(benzo[d][1,3]dioxol-5-ylmethylene)hex-2-enoic
acid with α- and nonprotein amino acids shown in Figure 1.
EPA is 4-ethyl substituted piperic acid. Piperic acid is
obtained from the hydrolysis of piperine, an alkaloid present in
black pepper (Piper nigrum-L/Piper longum). It has been
Pip-OH (BC06).
14
EPA was synthesized using the procedure described earlier.
3,3
3,3
β,β-disubstituted-β-amino acids, β -Ac c and β -Pip-OH,
(
6
15
were synthesized using a procedure reported in the literature.
10
Further, the conjugates of EPA, BC01−BC05, were synthesized
by treatment of EPA with ester hydrochloride of amino acids in
the presence of 1-(3-(dimethylamino)propyl)-3-ethyle carbo-
diimide hydrochloride (EDCI.HCl) and 4-methylmorpholine
11
reported that piperine suppresses tumor growth and metastasis
12
in 4T1 murine breast cancer model. The inhibition of
proliferation of human osteosarcoma by piperine via G2/M
phase arrest and metastasis by repressing MMP-2/-9 expression
Received: June 18, 2015
Accepted: August 31, 2015
13
has also been reported.
©
XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00257
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
NMM) in dry dichloromethane (Scheme S2, see Supporting
Letter
(
Information). The conjugate BC06 was synthesized by coupling
3,3
of valeryl-β Pip(NH)-NH-NH-Ph with EPA using EDCI·HCl
and NMM in dry dichloromethane (Scheme S3, see Supporting
Information).
The cell viability assay of all the conjugates was performed on
a panel of moderate to highly aggressive cancer cell lines. The
respective compounds were exposed to the cells with
concentration range (100 nM to 100 μM) for a period of 48
h. The conjugate BC06 exhibited its promising and consistent
cytotoxic activity in Panc-1 cell line with an IC concentration
5
0
of 4 μM, the lowest among all the conjugates, tested in all the
cell lines (Table 1).
Table 1. Cytotoxic Effects of Synthesized Compounds
(
BC01−BC06) on a Panel of Three Different Cancer Cell
Lines, viz., PANC-1, PC-3, and HCT-116 through Cell
Viability (MTT) Assay
Figure 2. Effect of conjugate BC06 on motility, colony formation and
invasive capability of pancreatic cancer cells. (A) Wound healing assay
was performed by treating PANC-1 cells with different concentrations
of conjugate BC06 to assess the degree of wound healing. Scratched
areas were photographed (20× magnification) at zero hour and at 24
a
PANC-1 IC50 (μM)
± SD
PC-3 IC50 (μM) HCT- 116 IC50 (μM)
± SD ± SD
compd
EPA
>100
>100 >100
h. (B) Colony formation assay was carried out against PANC-1 cells (1
BC01
BC02
BC03
BC04
BC05
BC06
6.7 ± 0.1
>100
43.5 ± 0.4
41.9 ± 0.3
16 ± 0.3
20 ± 0.2
45.2 ± 0.3
15 ± 0.1
47.1 ± 0.3
48.3 ± 0.5
49.8 ± 0.4
>100
3
×
10 cells/well) and treated with various concentrations of conjugate
BC06 along with DMSO for 5 days. The number of crystal violet
stained colonies were counted randomly and quantified, and images
were captured under inverted microscope at 20× magnification. (C)
Cells were treated with various concentrations of BC06 for 24 h and
analyzed for the invasive ability through matrigel invasion assay. The
invaded cells from five random fields in each treatment group were
counted and photographed under an inverted microscope (20×
magnifications). Data from three independent experiments were
subjected to statistical analysis. (n = 3, *p < 0.05).
42.2 ± 0.5
>100
10 ± 0.2
4 ± 0.5
47.6 ± 0.2
>100
a
Data were compared with untreated control, and IC50 values were
expressed as mean ± SD of three independent experiments.
As pancreatic cancer invasion and metastasis remains the
most critical determinant of resectability and hence survival, so
we aimed to study the effect of conjugate BC06 on the invasive
potential of PANC-1 cells in vitro. Wound healing assay was
performed to conclude whether conjugate BC06 could inhibit
motility of Panc-1 cells. After 48 h of incubation with 2.0 and
4
.0 μM of conjugate BC06, motility of PANC-1 cells inhibited
significantly (p < 0.05), similar to staurosporine (Figure 2A),
whereas the cells treated with vehicle DMSO essentially
migrated through the wounded area to close the wound
(Figure 2A). Also, the colony formation ability of Panc-1 cells
was decreased by conjugate BC06 in a statistically significant
manner (P < 0.05) (Figure 2B).
The critical event in tumor invasion and metastasis is the
ability of tumor cells to invade through the extracellular matrix,
allowing tumor cells to move beyond the limits of primary
tumor environment. Boyden chamber invasion assay was
carried out to study the effect of conjugate BC06 on cell
invasion. As shown in Figure 2C, treatment with BC06 (2.0 and
Figure 3. Effect of conjugate BC06 on invasion and matrix degradation
in PANC-1 cells. (A) PANC-1 cells were combined with neutralized
collagen, placed into the center of each well of a 96-well plate, and
allowed to solidify, forming a cell-collagen hemisphere with a distinct
boundary as described in materials and methods (Supporting
Information). Fresh media was then added to each well, and the
cells were allowed to invade into the surrounding matrix in the
presence or absence of indicated concentrations of conjugate BC06.
16
4.0 μM) inhibited cell invasion (P < 0.05). Further, the effect of
conjugate BC06 on cell invasion was observed using 3D
invasion assay. DMSO treated cells showed increased number
of invasive cells protruding beyond the original cell collagen
boundary, whereas indicated treatment of conjugate BC06
suppressed the cell invasion in a dose-dependent manner
(B) The antimetastatic effect of conjugate BC06 was assessed by
culturing PANC-1 cells on FITC conjugated gelatin matrix. Image-J
software was used to process and analyze the threshold areas of
degradation. Degradation zone was indicated by arrows. Statistical
analysis was performed on data obtained from three independent
experiments. (n = 3, *p < 0.05).
(
Figure 3A). These collectively demonstrate that EPA
3,3
conjugate of β -Pip-OH, BC06, alters the invasion and
metastatic potential of PANC-1 cells.
In cancer, metastasis and invasion are the two processes that
MMPs are thought to mediate. Type IV collagen acts as a
substrate for MMP-2 and MMP-9, and reports have connected
the overexpression of these enzymes in invasion and meta-
1
7
stasis. Cells were cultured on a cross-linked fluorophore
(FITC)-conjugated-gelatin matrix-coated coverslips for 24 h in
B
DOI: 10.1021/acsmedchemlett.5b00257
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
order to examine the ability of pancreatic cancer cells to
degrade the matrix. Figure 3B clearly shows that higher
concentrations of conjugate BC06 inhibited the matrix gelatin
degradation by aggressive Panc-1 cells (indicated by arrows).
Image-J software highlights the degraded area, which
supports the spots of gelatin matrix degradation. Further, we
ought to investigate the effect of conjugate BC06 on MMP-2
and MMP-9 expression in PANC-1 cells. The immunoblot
experiments demonstrated that treatment of PANC-1 cells with
conjugate BC06 negatively regulated both MMP-2 and MMP-9
expressions. As TIMP-1 is known to inhibit matrix metal-
loproteinases and seen to suppress metastasis, we further
studied the effect of conjugate BC06 on TIMP-1 expression.
The Western blot experiments demonstrated that the treatment
of conjugate BC06 resulted in upregulation of TIMP-1
expression in a dose-dependent manner (Figure 4A).
NF-KB in a dose-dependent manner (Figure 4B). As Akt is
24
known to regulate NF-KB via mTOR, we studied the effect of
conjugate BC06 on mTOR and S6K. The immunoblot results
demonstrated that conjugate BC06 treatment caused a sharp
decrease in phosphorylation of mTOR along with down-
regulation of S6K. There was no effect on total mTOR
expression following BC06 treatment (Figure 4C).
Collectively, we have shown here that the conjugate of EPA
3,3
with β -Pip-OH, BC06, significantly decreases invasion and
metastasis in PANC-1 cells by downregulating MMP-2 and
MMP-9 via suppression of PI3K/Akt/NF-kB pathway. Hence,
18
3,3
we strongly suggest that EPA conjugate with β -Pip-OH can
be used as an effective antimetastatic agent against advanced
pancreatic cancer.
ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00257.
Detailed procedure for the synthesis of EPA and
conjugates BC01-06, analytical data, and biological assays
(PDF)
AUTHOR INFORMATION
Corresponding Authors
E-mail: raj@iiim.ac.in.
E-mail: agoswami@iiim.ac.in.
■
*
*
Author Contributions
∥
H.A., N.A.W., and S.F. contributed equally to this work.
Funding
The work was supported by the Council of Scientific and
Industrial Research (CSIR), India under Network Project BSC-
120. The Institutional publication number is IIIM/1805/2015.
Figure 4. Conjugate BC06 inhibits MMP-2 and MMP-9 expression
through the inhibition of PI3K/Akt/NF-KB pathway. PANC-1 cells
were seeded in six well plates and treated with indicated
concentrations of conjugate BC06. Whole cell lysates were checked
for the expressions of indicated proteins by Western blotting. Values
below the Western blots indicate the relative protein expression
obtained by densitometric analysis of the bands.
Notes
The authors declare no competing financial interest.
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