RESEARCH ARTICLE

Dual Role of Par-4 in abrogation of EMT and switching on Mesenchymal to

Epithelial Transition (MET) in metastatic pancreatic cancer cells†

Archana Katoch a, b, Sujit Suklabaidya c ,Souneek Chakraborty a, b, Debasis Nayak a, b,

Reyaz Ur Rasool a, b, Deepak Sharma d, Debaraj Mukherjee d, Mir Mohd Faheem b, Anmol

Kumar e, Parduman Raj Sharma b, Shantibhusan Senapati c, Lekha Dinesh Kumar e, and

Anindya Goswami a, b, *

a
Academy of Scientific & Innovative Research (AcSIR), New Delhi, India.
b
Cancer Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road,
Jammu, Jammu and Kashmir - 180001, India.
c
Tumor Microenvironment and Animal Models Lab, Institute of Life Sciences (ILS), Nalco
Square Bhubaneswar, Orissa-751023, India
d
Natural Product Chemistry, Indian Institute of Integrative Medicine (CSIR), Canal Road,
Jammu, Jammu and Kashmir - 180001, India
e
Cancer Biology Division, Center for Cellular and Molecular Biology (CCMB), Uppal Road,
Hyderabad, Telangana - 500007, India

†
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
[10.1002/mc.22828]

Additional Supporting Information may be found in the online version of this article.

Received 24 November 2017; Revised 10 April 2018; Accepted 17 April 2018

Molecular Carcinogenesis
This article is protected by copyright. All rights reserved
DOI 10.1002/mc.22828

This article is protected by copyright. All rights reserved

  

*Corresponding Author
Dr. Anindya Goswami
Cancer Pharmacology Division,
Indian Institute of Integrative Medicine (CSIR),
Jammu Tawi - 180001, India
Telephone: 0191-2569111
Fax: 0191-2569333
Email: agoswami@iiim.ac.in

This article is protected by copyright. All rights reserved

  

ABSTRACT

Epithelial-mesenchymal transition (EMT), a critical event that occurs during invasion and

metastatic spread of cancer cells. Here, we conceive a dual mechanism of Par-4-mediated

inhibition of EMT and induction of MET in metastatic pancreatic cancer cells. First, we

demonstrate that 1,1’-β-D-glucopyranosyl-3,3’-bis(5-bromoindolyl)-octyl methane (NGD16), an

N-glycosylated derivative of medicinally important phytochemical 3,3’-diindolylmethane (DIM)

abrogates EMT by inducing pro-apoptotic protein Par-4. Induction of Par-4 (by NGD16 or

ectopic overexpression) strongly impedes invasion with inhibition of major mesenchymal

markers viz. Vimentin, Twist-1 and induction of epithelial marker- E-cadherin. Further, NGD16

triggers MET phenotypes in pancreatic cancer cells by augmenting ALK2/Smad4 signaling in a

Par-4-dependent manner. Conversely, siRNA-mediated silencing of endogenous Par-4 unveil

reversal of MET with diminished E-cadherin expression and invasive phenotypes. Additionally,

we demonstrate that intact Smad4 is essential for Par-4-mediated maintenance of E-cadherin

level in MET induced cells. Notably, we implie that Par-4 induction regulates E-cadherin levels

in the pancreatic cancer cells via modulating Twist-1 promoter activity. Finally, in vivo studies

with syngenic mouse metastatic pancreatic cancer model reveal that NGD16 strongly suppresses

metastatic burden, ascites formation, and prolongs the overall survival of animals effectively.

This article is protected by copyright. All rights reserved

Key words: Mesenchymal-epithelial transition, Par-4, NGD16, ALK2, Smad4, metastasis

This article is protected by copyright. All rights reserved

  

Abbreviations: EMT, epithelial-mesenchymal transition; MET, mesenchymal-epithelial

transition; Par-4, prostate apostsis response 4; PDAC, pancreatic ductal adenocarcinoma;

NGD16, 1,1’-β-D-glucopyranosyl-3,3’-bis(5-bromoindolyl)-octyl methane; DIM, 3,3’-

diindolylmethane; BMP7, Bone morphogenetic protein 7; TGF-β, Transforming growth factor β.

Introduction

Cancer remains to be one of the leading challenges for the health care systems across the globe.

The incidence of cancer is increasing at a tremendous rate and so is the morbidity and mortality

associated with it. Of the total deaths caused by cancer, the majority are attributable to cancer

metastasis [1,2] .The metastasis event occurs via a series of changes in the cancer cell; one such

being Epithelial to Mesenchymal Transition (EMT), a trans-differentiation process where the

epithelial cells lose their characteristic morphology and attain a mesenchymal phenotype. The

role of EMT has been implicated in various pathological as well as physiological conditions

including embryogenesis, tissue remodeling, tumor metastasis etc [3-5]. EMT is distinguished by

changes in cell morphology [6,7] downregulation of epithelial markers like E-cadherin, ALK2 as

well as the upregulation of mesenchymal markers, such as Vimentin, Twist-1 and Zeb-1 etc.

Mesenchymal to Epithelial Transition (MET), considered to be the inverse process of EMT,

prevents metastatic dissemination of cancer cells [8]. Although, contrary to EMT, the

mechanisms underlying the MET have been poorly understood, the re-expression of E-cadherin

is an important hallmark of MET [9]. A detailed understanding of this process will surely open

new avenues for circumventing cancer metastasis.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta

(TGF-β) superfamily and constitute a diverse, evolutionarily conserved family of secreted

signaling molecules critical for various developmental processes [10,11]. BMP7, another

This article is protected by copyright. All rights reserved

  

member of the BMP family, is known to counteract TGF-β1–induced EMT in developmental

stages [12-14]. ALK2, also termed ACTRI, is an activin type I receptor that mediates responses

for BMP7 [15]. Many findings suggest that BMP7 can induce MET possibly by ALK2,

phosphorylation of Smad 1, 5, and 8; and inhibition of EMT regulators viz Slug, SMA, Twist-1,

and Snail [16,17]. ALk2 phosphorylates Smad1/5/8, which then associates with Smad4 [18].

Smad4, also known as DPC4 (deleted in pancreatic carcinoma, locus 4), is an established tumor

suppressor frequently lost in pancreatic cancer [19,20]. Smad4 regulates transcription of target

genes through direct binding to DNA, interaction with other DNA-binding proteins and

recruitment of transcriptional coactivators and/or corepressors. Smad4 is reported to induce E-

cadherin in colon carcinoma cells as well as suppressed angiogenesis to impede tumor growth

[21,22]. Furthermore, low SMAD4 alterations was associated with long-term survival in patients

with of pancreatic adenocarcinoma [23]. Given the tumor suppressing role of Smad4, targeting

the ALK2/Smad4 signaling pathway, therefore, holds significant potential in controlling cancer

progression.

Prostate apoptosis response 4 (Par-4), a pro-apoptotic protein associated with tumor

suppressing activities, is highly expressed in cells undergoing apoptosis [24]. However, Par-4 is

either down-regulated or inactivated in many cancers [25-29]. Par-4 induction or stimulation by

diverse therapeutic agents/small molecules (doxorubicin, 3-AWA, arylquins etc.) could,

therefore, aid in development of improved anti-cancer therapeutics [30-32] In our recent report;

we have established that induction of intracellular Par-4 abrogates EMT and invasion in prostate

as well as breast cancer cells through modulation of oncogenic β-catenin [33].

Extracellular/secreted Par-4 proteins also have the capability to block MMP-2-mediated invasion

and angiogenesis in human cervical cancer (HeLa) as well as in prostate cancer (PC-3) cells [31].

This article is protected by copyright. All rights reserved

  

Hence, Par-4 is a potential therapeutic target for the discovery of small molecule modulators to

curb invasion and metastasis of aggressive cancer cells.

3,3’-Diindolylmethane (DIM) is a well-documented bioactive constituent present in

cruciferous vegetables which are known for various health benefits [34,35]. A plethora of

research has demonstrated the role of DIM in abrogating breast and prostate cancer through

several mechanisms, including induction of apoptosis, execution of oxidative stress responses,

balancing estrogen metabolism, cell cycle arrest, and other anti-proliferative mechanisms [36-

42]. These properties of bis-indoles have attracted the researchers across the globe to develop

them as potent anti-cancer and anti-metastasis compounds. Furthermore, DIM is also reported to

induce the expression of Prostate apoptosis response 4 (Par-4) [43]. Moreover, in one of our

previous studies, we have established that NGD16 [1,1,-β-D-glucopyranosyl-3,3’-bis(5-

bromoindolyl)-octyl methane [44], a potential N-glycosylated derivative of 3,3’,-

diindolylmethane, blocks angiogenesis by targeting the glucose regulated protein, 78 kDa

(GRP78) [45]. Although angiogenesis is a crucial phenomenon that facilitates invasion and

metastasis of tumor cells, rationally, in this study, for the first time we have reported the MET-

inducing ability of NGD16 exerted through Par4-mediated inhibition of EMT and invasion in

metastatic pancreatic cancer cells.

Materials and Methods

Cell Culture and Transfection

The cells lines viz MIA Paca-2, Panc-1, HT-29, HCT-116 and BxPC-3, used in this study were

originally obtained from European Collection of Cell Culture (ECACC). MIA Paca-2 and Panc-1

cells were cultured in DMEM; BxPC-3 and HT-29 in RPMI 1640; HCT-116 in McCoy’s media,

supplemented with 10% FBS, penicillin and streptomycin. Cells were incubated at 37 ○C in a

This article is protected by copyright. All rights reserved

  

humidified atmosphere of 5% CO2 incubator (Galaxy170R, New Brunswick, Germany). Sub-

culturing was done when 60–70 % of confluence was reached to ensure cells in a healthy

condition. siRNA Knockdown Assay was carried out using lipofectamine 3000 transfection kit

(Invitrogen) while the GFP and GFP-Par-4 transient transfections were performed with

DharmaFECT-1transfection reagent or Neon Transfection System (MPK5000,Invitrogen,

Carlsbad, CA) according to manufacturer’s protocol. The UN-KC-6141 mouse pancreatic cancer

cell line was a generous gift from Dr. S. K. Batra at the University of Nebraska Medical Center,

USA to the laboratory of Dr. S. Senapati at Institute of Life Sciences (ILS), Bhubaneswar, India.

The cells were obtained through material transfer agreement and were maintained as reported

earlier [46].

Chemicals, Antibodies, and DNA Construct

RPMI-1640, DMEM and fetal bovine serum (FBS) were obtained from Invitrogen.

Streptomycin, Penicillin G, Trypsin-EDTA, Phenylmethylsulfonyl fluoride (PMSF), Bradford

reagent, Paraformaldehyde, Phosphate buffer saline, Protease inhibitor cocktail,

Dimethylsulphoxide (DMSO), and Triton X-100 used in the study were from Sigma chemicals

(St. Louis, MO). NGD16 was obtained from our Natural Plant Chemistry division, IIIM, Jammu

and dissolved in DMSO [44]. The final concentration of DMSO in the experiment never

exceeded 0.2%. Antibodies for E-cadherin, Vimentin, Twist-1, Par-4, NF-kB (p65), Sox-2,

ALK2 and Smad4; UltraCruz DAPI (4,6-diamidino-2-phenylindole) mounting medium were

obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Beta-actin, Anti-rabbit and anti-

mouse secondary antibodies; small interfering RNA (siRNA) oligonucleotide duplexes targeted

against human Par-4 were obtained from Sigma chemicals. GFP and GFP-Par-4 were generous

gifts from Dr. Vivek Rangnekar (University of Kentucky, Lexington, Kentucky).

This article is protected by copyright. All rights reserved

  

Cell Lysate Preparation and Western Blotting

The standard procedure was followed for cell lysis and immunoblotting [33]. In brief, cells were

subjected to their respective treatment. Post-treatment, cells were collected, centrifuged and

added with lysis buffer. The cell lysates were collected by centrifugation. Protein concentration

was quantified and equal amount of proteins from each sample was subjected to western blot

analysis. The proteins were separated by SDS–PAGE, transferred onto PVDF membrane

(Millipore, Billerica, MA), blocked with 5% skim milk and subsequently incubated with the

specified antibodies, either at room temperature for 3 h or overnight at 4 ○C. Thereafter, the blots

were washed and probed with the respective secondary antibodies coupled with horseradish

peroxidase. After thorough washing, protein bands were visualized using enhanced

chemiluminescence reagent (Millipore) and captured onto Biomax light film (Eastman Kodak

Co., Rochester, NY).

Matrigel Invasion Assay (Boyden Chamber Assay)

The effect of NGD16 on cell invasion was determined using BD Biocoat Tumor Invasion Assay

System (BD Bioscience, Bedford, MA) following the instructions of the manufacturer. Briefly,

Panc-1 and MIA Paca-2 cells (1.2 X 106) cells were cultured in the presence of different

concentrations of NGD16 or vehicle (DMSO) for 48 h in the upper chambers/inserts in the

presence of serum-free media, and the bottom wells were filled with chemoattractant (complete

media with 10% FBS). Cells were allowed to migrate at 37 ○C. After 48 h, the Matrigel-coated

polycarbonate filters were taken out, the non-migrating cells were removed from the upper

chamber with a cotton swab and the insert was fixed with methanol and stained with 0.1 %

crystal violet solution. For each replicate (n=3), migration of the cells was quantified by counting

the stained cells (cells per five fields) under an inverted microscope.

This article is protected by copyright. All rights reserved

  

In Situ Fluorescent Gelatin Degradation Assay

Cross-linked-fluorophore (FITC)-conjugated gelatin matrix-coated coverslips were prepared as

described [47]. Gelatin-coated coverslips were quenched with RPMI containing 10% fetal bovine
○
serum at 37 C for 60 mins prior to plating cells. To assess the formation of

podosomes/invadopodia and degradation of FITC-gelatin matrix, Panc-1 cells were cultured on

FITC gelatin-coated coverslips. After 16 h, cells were treated with 2 µM of NGD16, in presence

or absence of TGF-β1, for 48 h. The TGF-β1 treated Panc-1 cells were used as a positive control

for gelatin degradation. The cells were then stained using the DAPI mounting media and

observed under FLoid Cell Imaging Station for determination of matrix gelatin degradation. The

threshold area of degradation was quantified by Image-J software.

Immunocytochemistry

For immunocytochemistry, the previously published protocol was followed with some

modifications [33]. Briefly, Panc-1 and MIA Paca-2 cells were seeded, at a density of 3x104

cells/well, in an eight-well chamber slide and then subjected to the respective treatment for 48 h.

Following treatment, the cells were washed thrice with PBS, fixed with 4% paraformaldehyde

(pH=7.4) for 15 mins, permeabilized with 0.1-0.3% Triton X-100(0.1% for E-cadherin and 0.3%

for Vimentin) for 10 mins and then blocked with 1% bovine serum albumin for 30 mins.

Consequently cells were immunostained with anti-E-cadherin, anti-Vimentin, anti-Par-4 and

anti-Smad4 antibodies at a dilution of 1: 100 at 4 ○C overnight. Further, the incubated samples

were washed with PBS six times, five min each and thenceforth probed with anti-rabbit Alexa

Fluor-488 (anti-E-cadherin and anti-Par-4)/anti-mouse Alexa Fluor-488 (anti-Smad4)/ anti-rabbit

Alexa Fluor-555 (anti-Vimentin) conjugated secondary antibody at a dilution of 1:500 for 1 h at

room temperature in the dark. Next, the cells thoroughly washed with PBS and counterstained

This article is protected by copyright. All rights reserved

  

with DAPI containing mounting medium in the dark. The slide was mounted with a coverslip,

sealed and images were captured with Floid Cell Imaging Station (Life Technologies) at 20X

magnification.

Scratch Motility (Wound Healing) Assay

The assay was performed according to the previously published protocol [31]. Cells were plated

at a density of 5x105 cells/well in a six-well plate, incubated to form of a confluent monolayer,

and afterward, serum starved for 24 h. These Confluent monolayers of cells were then scratched

by a sterile pipette tip and washed with serum-free medium to remove detached and floating cells

and photographed (time 0 h). The cells were subjected to varying concentrations of NGD16 for

24 h in the presence of medium containing 1% serum. The cell migration images were taken

using Nikon D3100 inverted microscope camera (20X magnification) and the percentage of

wound closure was also determined.

Cell Viability Assay

The effect of NGD16 on cell viability was determined against a panel of cancer cell lines viz:

Panc-1, MIA Paca-2, BxPC-3, HT-29 and HCT-116 through MTT assay. Briefly, cells were

seeded in 96 well tissue culture plates (Nunc) at a density of 3 × 103 cells per well and treated

with varying concentrations of NGD16 in triplicates. DMSO was added as a vehicle and the final

concentration of the DMSO was maintained at 0.2 % in the culture medium. After 44 h of

incubation, MTT dye solution (2.5 mg/ml) was added into the medium and cells were incubated

for another 4 h at 37 °C in 5 % CO2. The amount of coloured formazan derivatives formed was

measured by taking optical density (OD) using a microplate reader (TECAN, Infinite M200 Pro)

This article is protected by copyright. All rights reserved

  

at 570 nm and the percentage cell viability was calculated. The IC50 values were calculated using

GraphPad Prism software (Version 5.0).

Cell Scattering Assay

The assay was performed following the previously published protocol [48]. Briefly, Panc-1 cells

at a density of 1000 cells/well were seeded in 6-well plates and incubated for 3–4 days to form

small, discrete colonies. VEGF (20 ng/ml) was then added to the growth media to stimulate these

cells, in the presence and absence of various concentrations of the NGD16. After 48 h of

incubation, colonies were fixed, stained with 0.2% crystal violet and the photographs were

captured.

Smad4/Par-4 siRNA Knockdown Studies

Panc-1/MIA Paca-2 cells were seeded in a 6-well plate (for Western blotting) or eight-well

chamber slide (for immunocytochemistry) and transiently transfected with Par-4/Smad4 siRNA

using lipofectamine 3000 or Neon Transfection System (MPK5000, Invitrogen, Carlsbad, CA)

according to manufacturer’s protocol. After 24-36 hours, the cells were treated with the

respective NGD16 concentrations and accordingly subjected to Western blotting or

immunocytochemistry.

Luciferase Reporter Assay

Twist-1 reporter construct (PGL3-Twist-1-luc), a kind gift from Dr. Mien-Chie Hung, MD

Anderson Cancer Center, Texas, was employed to measure the Twist-1 transcription activity using a

luciferase reporter assay system. Panc-1 cells were transfected with Twist-1-luc/PGL3 vector

backbone and PGL3 was used as the respective control, with and without NGD16 (3µM). Within 48

h, the same cells were transfected with Renilla construct. The firefly luciferase and Renilla

luciferase activity were analyzed by two step process using the Dual Glo reagents (E2940, Promega,

This article is protected by copyright. All rights reserved

  

Madison, USA) according to the manufacturer’s instruction. The final results were based on three

independent experiments.

In Vivo Studies for Metastasis

All animals used in this study were bred and maintained at the central animal facility of Institute

of Life Sciences, Bhubaneswar, India. Animals were maintained at 20-25 °C in a 12 h light dark

cycle, routinely monitored for their diet and water consumption and proper sanitations were

maintained to avoid any risk of possible pathogenic contamination. Animal studies were

performed in accordance with the experimental guidelines that were approved by the Institutional

Animal Ethics Committee. During the animal experiments, special handling and care was taken

in a humane way, so that no extra pains/injuries were imparted to the animals. To minimize the

mortality of animals during experimentation, only a limited number of animals were employed to

yield the statistically significant results. To evaluate the in vivo anti-metastatic efficacy of

NGD16, UN-KC-6141 syngenic mouse metastatic pancreatic cancer model was prepared.

Accordingly, six week old healthy male C57BL/6 mice (body weight: 25 – 30 g) were taken and

acclimatized for 2-3 days. For the orthotopic implantation of pancreatic tumor cells, animals

were first anaesthetized with intraperitoneal administration of a cocktail mixture of ketamine (80

mg/kg) and xylazine (8 mg/kg). After sterilizing the incision site, approximately 1 cm long

incision was made in the abdomen near the position of spleen. The pancreas along with the

spleen was gently pulled up to the incision site and 0.5 × 106 UN-KC-6141 cells in 50 µl PBS

were injected into the pancreas using a 30-guage needle. After resetting all the pre-moved

organs, the abdomen was closed with absorbable catgut sutures and the skin incision was closed

with interrupted sutures of non-absorbable material. The animals were monitored regularly and

the skin suture material was removed at approximately five days after the surgical procedure.

This article is protected by copyright. All rights reserved

  

Three days after injecting cancer cells, all the tumor-bearing animals were randomly divided into

two groups (n = 5 per group). The formulation of NGD16 was prepared by dissolving the

compound in DMSO, chremophor, and PBS in a ratio of 0.5: 0.5: 9.0. The formulation was

injected intraperitoneally into the animals at a pre-standardized dose of 30 mg/kg, b.w (final

volume 200 µL) in each alternate day for twenty days. The animals in control group received

same volume of vehicle containing a mixture of DMSO, chremophor, and PBS. On the 22 nd day,

all animals were sacrificed and gross necropsy was performed to determine the extent of

metastasis. The tumor tissue samples were homogenized and later subjected to immunoblotting

to check the expression of Par-4.

Statistical Analysis

Data were expressed as mean ± S.D. of at least three independent experiments performed,

analyzed by Student’s t-test and *P < 0.05 values were considered significant in all cases. IC50

values were determined with the help of GraphPad Prism software Version 5.0 (GraphPad

Software, La Jolla, CA, USA) by taking the log of inhibitor versus response.

Results

NGD16-mediated inhibition of EMT and induction of MET abrogates invasion in

pancreatic cancer cells

EMT is an important event that results in the transition of an epithelial cancer cell to a

transforming mesenchymal phenotype which ultimately leads to tumor growth and metastasis

[3,4]. Physiologically, the reverse mechanism of EMT is known as mesenchymal to epithelial

transition (MET). Diindolylmethane (DIM) scaffolds are demonstrated for abrogation of EMT in

diverse cancer models but yet to be explored for MET induction. The medicinal chemistry group

of our laboratory synthesized and reported some novel derivatives of DIM with potential

This article is protected by copyright. All rights reserved

  

anticancer activities and by SAR analysis NGD16 (previously reported as compound 7d) was

found to be the most active compound against a panel of cancer cell lines [44]. Cell viability

assay results unveiled that the IC50 values of NGD16 in Panc-1, MIA Paca-2, HCT-116, HT-29

and BxPC-3 were found to be 2.5, 2.8, 3.7, 2.9 and 3.4 µM respectively [45] (Supplementary

Table S1). We investigated the effect of NGD16 on markers of MET and EMT in metastatic

pancreatic cancer cells through immunoblotting wherein the results demonstrated that the

expressions of epithelial markers such as E-cadherin and ALK2 were augmented consistently.

However, we found a sharp decline in the mesenchymal markers viz Vimentin and Twist-1 in a

dose-dependent manner in Panc-1 and MIA Paca-2 cells (Fig. 1A). Since EMT is reported to

promote stemness in the cancer cells [49], we also evaluated the effect of NGD16 on the

stemness factor ,Sox2. The Sox2 protein levels were found to decrease gradually. We then

assessed the time-dependent effects of NGD16 on these markers in Panc-1 and MIA Paca-2. The

western blot analysis data demonstrated a robust increase in the expression of E-cadherin and

ALK2 along with decrease in Vimentin, Twist-1 and Sox2 protein levels at 48 h (Fig. 1B).

It is well-known that during EMT cells switch to a mesenchymal phenotype conferring a loss of

cell-to-cell adheren protein E-cadherin along with an extension of Vimentin cytoskeleton. Hence,

we ought to study the expression of Vimentin and E-cadherin in Panc-1 cells in the presence of

NGD16 by immunocytochemistry. Remarkably, the data illustrated a fair reduction in Vimentin

expression (Fig. 1C) in NGD16-treated Panc-1 cells compared to the vehicle; coherent with the

western blotting results. However, the expression of E-cadherin, the hallmark of MET, was

found to be significantly upregulated under similar conditions.

Since EMT triggers invasion and migration properties of transformed cells, we were

interested in investigating the effect of NGD16 on cell invasion. The Boyden chamber invasion

This article is protected by copyright. All rights reserved

  

assay was carried out to determine the ability of NGD16-treated Panc-1 and MIA Paca-2 cells to

invade through biological matrices in vitro. As shown in Figure 1D and E, at an increasing

concentration, NGD16 abrogated the invasion capability of Panc-1 and MIA Paca-2 cells

compared to the vehicle. The ability of Panc-1 cells to degrade the matrix in the presence of

NGD16 was also examined by culturing cells on a crosslinked fluorophore (FITC)-conjugated-

gelatin matrix-coated coverslips for 48 h. Figure 1F and 1G clearly demonstrated that NGD16

inhibited the TGF-β1 induced migration and matrix gelatin degradation of Panc-1 cells. Further,

the reduction in the threshold areas of degradation in the TGF-β1 (5 ng/mL) plus NGD16 (2 µM)

condition, obtained from Image-J software, as compared to the TGF-β1 treated control

underscored the anti-invasive effect of NGD16. The wound healing assay performed to assess

the effect on cell motility displayed a pronounced suppression of TGF-β1 migration in Panc-1

cells (Supplementary Figure 1A & 1B). Inhibition of cell scattering, a predominant feature of a

mesenchymal cell, was also studied (Supplementary Figure 1C). Next, we wanted to verify

whether global inhibition of EMT, as observed in the results above, always overlaps with MET

induction. For that, we treated the Panc-1 cells with two different EMT blockers, 3-

azidoWithaferin A (3-AWA) and DIM, and observed for any ALK2 induction. However, we

failed to notice increase in the ALK2 protein (Supplementary Figure 1D). All the above results

support that NGD16 reverses EMT and leads to MET induction, thereby, impeding invasion and

migration in Panc-1 and MIA Paca-2 cells.

NGD16 Induces MET by Modulating the ALK2/Smad4 Signalling

A plethora of recent reports demonstrate the ability of BMP7 in inhibiting EMT and initiating

MET via the upregulation of its receptor and MET associated marker, ALK2 [10]. Rationally, we

were interested to know whether NGD16-induced MET is actually a consequence of modulation

This article is protected by copyright. All rights reserved

  

of the BMP7 pathway or its effectors. It is established beyond doubt that Smad4 is an important

downstream signalling molecule in the BMP7 signaling. So, we also examined the effect of

NGD16 on Smad4 expression level. Surprisingly, upon treatment with increasing concentrations

of NGD16, a steady increase in the expression levels of E-cadherin and ALK2 was achieved in

Panc-1 and MIA Paca-2 cells (Fig. 2A) which was concommitant with a gradual increase in the

Smad4 protein levels. Panc-1 cells stimulated with BMP7, a known MET inducer, were taken as

the positive control and analysed for the subsequent increase in the ALK2 and Smad4 protein

levels (Fig. 2B). The immunocytochemistry analysis also validated the western blotting results

(Fig. 2C), depicting a significant amplification in the ALK2 signal in Panc-1 cells treated with 2

µM NGD16 compared to the vehicle.

Additionally, to verify significance of Smad4 in E-cadherin induction, we performed the same

set of experiments on HT-29 (Smad4+/-), BxPC-3 (Smad4+/-) and HCT-116 (Smad4+/+) cell lines.

While all the cell lines showed augmented levels of ALK2 upon treatment with varying

concentrations of NGD16, a gradual increase in the E-cadherin protein levels was exclusive to

HCT-116 cells (Smad4+/+) (Fig. 2D). The inability of HT-29 and BxPC-3 cells to upregulate E-

cadherin in response to NGD16 underscores that the mutated Smad4 (Smad+/-) background

resistant to E-cadherin upregulation. Furthermore, treatment of Panc-1 cells with NGD16

significantly inhibited the TGF-β1-induced EMT and gradually stimulated the MET phenotype

as evident by the changes in the cell morphology upon microscopic examination (Fig. 2E).

Moreover, NGD16 also countered the mesenchymal phenotype in the MIA Paca-2 cells

(Supplementary Figure 2A). Together these results highlight that NGD16 reverses the TGF-β1-

induced mesenchymal phenotype, induces MET by targeting the ALK2/Smad4 axis and that an

intact Smad4 background is indispensable for E-cadherin upregulation.

This article is protected by copyright. All rights reserved

  

NGD16-Mediated Amplification of E-cadherin is Smad4-Dependent

Since Smad4 gene is inactivated in about half of PDAC as well as one third of colorectal cancers

[50,51] and Smad4 was identified as a potential tumor suppressing gene, therefore, it can be

assumed that Smad4 must hold an effective role in maintaining epithelial traits of PDAC cells.

Moreover, Muller et al. [52] have shown that restoration of Smad4 in SW480 human colon

carcinoma cells, led to a spontaneous induction of functionally competent cadherins, with

restitution of cell to cell adhesion coupled with morphological reversion to an epithelial

phenotype. Therefore, we sought to examine the effect of knock down of endogenous Smad4,

using Smad4 siRNA, on E-cadherin expression in MIA Paca-2 and Panc-1 cells. Interestingly,

we observed that MIA Paca-2 cells treated with 3 µM NGD16 caused a marked upregulation in

the E-cadherin protein level (Fig. 3A, Lanes 4). However, this upregulation was drastically

hampered when the MIA Paca-2 cells were treated with NGD16 in presence of Smad4 siRNA

(Fig. 3A, Lane 5). Similar results were observed in the ICC of Panc-1 cells tranfected with

Smad4 siRNA in presence and absence of 2 µM NGD16. Depicting. Smad4 and E-cadherin

expressions were significantly diminished in the presence of siRNA Smad4 plus NGD16 wells

compared to alone NGD16 (Fig. 3B). Moreover, as shown in Fig. 3C, in the context of Smad4

background, E-cadherin protein levels were augmented (Honey-comb structure) in the NGD16
+/+
treated HCT-116 cell (Smad4 ) compared to the vehicle, whereas no such induction of E-
+/-
cadherin was observed in the NGD16 treated HT-29 cells (Smad4 ) (Fig. 3C). Of note, the

cell-cell adhesion as well as honey-comb structure was retained in HCT-116 cells but not in HT-

29 cells. Together these results demonstrate that the induction of E-cadherin observed due to

NGD16 treatment is majorly Smad4 (Smad4 +/+ )-dependent.

This article is protected by copyright. All rights reserved

  

Ectopic Par-4/NGD16 Induced Par-4 Negatively Regulates EMT

Recent studies have demonstrated the induction of PAR-4 by DIM and its derivatives. These

studies also implied the role of Par-4 in suppressing tumorigenesis, through the regulation of

various anti-apoptotic proteins [30,31]. Furthermore, the ability of PAR-4 in inhibiting EMT and

its regulators is now been well documented [33]. Rationally, we sought to examine whether there

was any effect of NGD16 on endogenous Par-4 levels. Interestingly, when MIA Paca-2 and

Panc-1 cells were subjected to varying concentrations of NGD16, indeed there was a marked

increase in the endogenous Par-4 levels in a dose-dependent (Fig. 4A) as well as time-dependent

manner (Fig. 4B), wherein maximum Par-4 expression was observed at 48 h. For further

confirmation, the Panc-1 and MIA paca-2 cells were subjected to 2 and 3 µM concentration of

NGD16 for 48h, respectively, along with the appropriate control to examine the Par-4 induction

through immunocytochemistry. Evidently, there was a robust induction of Par-4 in both Panc-1

as well as MIA Paca-2 cells upon exposure with NGD16 (Fig. 4C).

Further, in order to validate the Par-4-mediated inhibition of EMT, we transfected the Panc-1

cells with GFP/GFP-Par-4 plasmids and accordingly subjected them to immunoblotting and

immunocytochemistry. As depicted in Figure 4D and the respective bar-graph (Fig. 4E), when

GFP-Par-4 was overexpressed in Panc-1 and MIA Paca-2 cells, a considerable increase in E-

cadherin levels was achieved compared to GFP transfected cells . The ICC images clearly

revealed a robust attenuation of Vimentin expression in GFP-Par-4 transfected cells (Fig.

4F).Similar results were obtained from immunocytochemistry analysis of GFP-Par-4 transfected

MIA Paca-2 cells probed for Vimentin (Supplementary Figure 3). To further examine the effects

of Par-4 overexpression on invading ability of Panc-1 cells, we transfected the Panc-1 cells with

GFP/GFP-Par-4 and then performed tthe Boyden chamber assay. Interestingly, the invasion

This article is protected by copyright. All rights reserved

  

capability of the GFP-Par-4 transfected Panc-1 cells was markedly abrogated as compared to the

GFP-transfected cells (Fig. 4G & 4H). Collectively, these results imply that ectopic Par-

4/NGD16 induced endogenous Par-4 negatively regulates EMT markers and invasion of

pancreatic cancer cells.

NGD16-Induced MET is Par-4-Depenedent

The preceeding results indicating that NGD16 induced endogenous Par-4 levels as well as Par-4

overexpression severely abrogated the EMT markers, prompted us to further verify the

mechanism behind. We found that the gradual amplification of endogenous Par-4 level (due to

NGD16 treatment) conferred an inverse correlation with EMT markers viz. Twist-1, Vimentin

(Fig. 5A). Further, NF-κB is an important transcription factor that is crucial in many steps of

cancer initiation and progression where it cooperates with many other signaling molecules. Upon

treatment with NGD16, we also observed a significant abrogation in the NF-kB (p65) protein

levels [53,54]. This exciting direct correlation of Par-4 with E-cadherin, Smad4 and ALK2 was

further validated by over-expression of Par-4 indicating that upon transfection with GFP-Par-4,

the ALK2 and Smad4 levels were significantly induced as compared to the GFP-transfected

Panc-1 cells (Fig. 5B and Fig. 5C).

Rationally, we sought to investigate the effects of silencing of endogenous Par-4 on MET

markers. In order to do that, we transfected Panc-1 cells with Par-4 siRNA and probed for the

various MET markers. The results showed a complete disappearance of ALK2 and Smad4 in

NGD16 and siPar-4 lane compared to Vehicle/scramble siRNA lanes (Fig. 5D, Lane 4 and 5).

However, in presence of siPar-4, the NF-kB (p65) expression was found to be upregulated (Fig.

This article is protected by copyright. All rights reserved

  

5D, Lane 3 and 5) compared to the NGD16-treated condition underscoring the parallel role of

Par-4 in the regulation of NF-kB. To validate that the previously observed Smad4-mediated E-

cadherin induction (due to NGD16 treatment) was actually Par-4-dependent, the Par-4

knockdown studies were performed in Panc-1 cells and the cells were probed with E-cadherin for

immunocytochemistry. The results clearly illustrated that NGD16-induced E-cadherin expression

was abrogated in the presence of siRNA Par-4 (Fig. 5E).Collectively, these above results implied

that the ALK2, Smad4 and E-cadherin, activation upon NGD16 treatment was primarily Par-4

dependent. Furthermore, we sought to examine whether the induced Par-4 showed any relevant

effects on major transcription factors regulating E-cadherin. Since, Twist-1, a helix loop helix

transcription factor is responsible for controlling E-cadherin by binding to its promoter, we

hypothesized that Par-4 might be regulating Twist-1 promoter activity.For confirmation, we

transfected Panc-1 cells with Twist-1 luciferase construct along with PGL3 as a suitable control.

These cells were then subjected to 2 µm Vehicle/NGD16 or transfected with GFP/GFP-Par-4.

After 48 h of incubation, the luciferase activities within the cells were analyzed using the dual

glow luciferase assay system. The vector transfected cells showed comparatively more luciferase

activity. The luciferase activity got significantly decreased where the Twist-1 luciferase

construct-transfected Panc-1 cells were treated with NGD-16 compared to the vehicle Vehicle

(Fig. 1F). Similarly, the GFP-Par4 transfected displayed lessened luciferase activity than the

GFP transfected cells with the Twist-1 luciferase construct. All these results, validate that the

NGD-16 induced MET via ALK2, Smad4 and E-cadherin upregulation and attenuation of EMT

is Par-4 depentent.

This article is protected by copyright. All rights reserved

  

NGD16 Abrogates Metastasis in Syngenic Mouse Metastatic Pancreatic Cancer Model

To investigate the effect of NGD16 on the metastatic abilities of UN-KC-6141 aggressive

pancreatic cancer cells in vivo, the tumor-bearing animals were treated with NGD16 (30 mg/kg

b.w.) every alternate day for 20 days. Interestingly, the gross necropsy analysis of different

organs such as peritoneum, liver, spleen, kidney, and mesentery unveiled the presence of

substantial quantities of visible metastatic tumor nodules in vehicle treated group. Conversely,

the metastatic tumor burden was decreased significantly in the NGD16 (30 mg/kg b.w.) treated

group compared to the vehicle (Fig. 6A). Especially, there found 66.7% inhibition in metastasis

incidence to the liver, 50% inhibition of metastasis each into the peritoneum and kidney, and

33.4% inhibition into the mesentery in NGD16 treated group of animals, which indicates the

strong anti-metastatic effect of the compound (Fig. 6B). Further, the level of ascites was reduced

considerably (40%) in NGD16 treated group of mice compared to the vehicle treated group (Fig.

6C). Moreover, NGD16 greatly improved the overall survival of the animals; at day 16 one

animal and on day 20 two animals died from the vehicle treated group, whereas, in NGD16

treated group only one animal died at day 20 (Fig. 6D). Furthermore, the immunoblots of the

tumor tissue lysates and the respected bargraph showed increased Par-4 expression in the tumor

tissues obtained from NGD16 injected mice as compared to control (Fig. 6E). These results

together demonstrate that NGD16 is a potential anti-metastatic lead molecule that can curb

severe metastatic burden, level of ascites formation, and progress the overall survival of animals

with metastatic pancreatic cancer.

Discussion

3,3’-Diindolylmethane (DIM) is a major chemopreventive agent found in leafy cruciferous

vegetables (Broccoli, cauliflower etc.). It is a desirable drug candidate with diverse

This article is protected by copyright. All rights reserved

  

pharmacological properties including, anti-invasive, anti-angiogenic and immunomodulatory

activities [36-42]. The consumption of leafy cruciferous vegetables is recommended by

physicians because of its profound anti-oxidant properties. In this study, we have demonstrated

that NGD16, a potential derivative of DIM has consistently shown MET inducing property in

Panc-1 and MIA Paca-2 cells in a dose and time-dependent manner by modulating the

BMP7/ALK2/Smad4 axis (Fig. 7). Significantly, the TGF-β1-induced EMT in Panc-1 cells was

completely blocked upon treatment with NGD16.

The role of Bone morphogenetic proteins (BMPs) is well known to counteract TGF-β1-induced

EMT in developmental stages [12-14] and positively related to the expression ratio of E-cadherin

and Vimentin, suggesting the role of BMP7 in MET induction [55,56]. ALK2 upregulation is

reported to be a significant event in the BMP7-induced MET [57]. Interestingly, NGD16 caused

a gradual increase in the protein levels of ALK2 in both Panc-1 and MIA Paca-2 which were

positively related to the E-cadherin upregulation and inversely related to the pro-EMT markers

viz. Vimentin and Twist-1. Smad4 is an important tumor suppressor and a crucial downstream

player in the BMP7-ALK2-Smad4 axis. Growing evidences suggest that MET induced by BMP7

could abrogate prostate cancer bone metastasis, breast cancer, bone metastasis, and renal fibrosis

[13,55,56]. BMP7 also impedes tumor growth of human uveal melanoma in vivo [58].

Interestingly, we found that treatment of pancreatic cancer cells with NGD16 caused a robust

amplification of Smad4 that corresponded with the increased E-cadherin protein levels. For the

context of MET induction, BMP7/ALK2 signaling plays a pivotal role to sustain/elevate cellular

E-cadherin level. However, upon EMT onset, Snail transcriptionally represses E-cadherin but

BMP7 confers a strong antagonistic role on Snail to maintain the E-cadherin level [59].

This article is protected by copyright. All rights reserved

  

Therefore, negative regulation of Snail transcription factor holds a high cellular integrity to

maintain epithelial traits.

Basically, the induction of MET corresponds with the typical phenotypic changes in cellular

morphology as well as their intrinsic signaling mechanism governing cellular motility. A large

body of literature shows that downregulation of E-cadherin; upregulation of Vimentin and Twist-

1 are associated with aggressive tumor growth, local invasion and metastasis to distant organs in

many cancers [60,61]. Therefore, Par-4 mediated suppression of Twist 1 is paving a stone for its

(Par-4) anti-metastatic role apart from tumor suppression. However, in our experimental set up,

we found a drastic drop in Vimentin expression as well as negative regulation of Twist-1

promoter in the cells with over-expressed GFP-Par-4/induced Par-4 indicating that a

stoichiometry in Par-4 levels could be a decisive factor for EMT modulation. Although, it would

not be overambitious to suggest that the induced Par-4 mediated inhibition of NF-κB might have

negative impact on the master regulator Snail which consequently regulates Vimentin. On the

other hand, NF-κB directly upregulates both Twist-1 and Twist-2 expression to prevent cells

from undergoing apoptosis [62]. Interestingly, Twist-1 can transcriptionally repress E-cadherin

promoter activity by directly binding to the proximal E-boxes 2 and 3 in the E-cadherin promoter

[63].

Nutshell, the induction of Par-4, either by chemotherapeutics or exogenous source could be

potentially exploited for the development of anti-metastatic therapy. Since, Par-4 is a

physiologically relevant protein and expressed in almost all the vertebrate organs, therefore, the

implication of Par-4 induction in Smad4+/+ background poses enormous prospect to dually

control the severity of aggressiveness of PDACs through blocking of EMT and simultaneously

boosting MET properties in these cells (Fig. 7). Thus, NGD16 is a promising candidate to curb

This article is protected by copyright. All rights reserved

  

metastasis of PDAC and simultaneously enhances the endogenous Par-4 levels in pancreatic

cancer cells.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

The work was supported by Department of Biotechnology, Government of India, Grant No.

BT/PR6768/MED/30/893/2012 to AG. We thank our Director Dr. R.A. Vishwakarma for

encouraging us to accomplish this work. We are grateful to Dr. V. M. Rangnekar (University of

Kentucky, USA) for providing GFP-Par-4 construct. The authors are grateful to Department of

Biotechnology, Govt. of India, and Council of Scientific and Industrial Research (CSIR) for

providing fellowship to the research scholars.

This article is protected by copyright. All rights reserved

  

References

1. Gupta GP, Massagué J. Cancer metastasis: building a framework. Cell 2006;127(4):679-

695.

2. Steeg PS. Tumor metastasis: mechanistic insights and clinical challenges. Nat Med

2006;12(8):895-905.

3. Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer

2002;2(6):442-454.

4. Moustakas A, Heldin C-H. Signaling networks guiding epithelial-mesenchymal

transitions during embryogenesis and cancer progression. Cancer Sci 2007;98(10):1512-

1520.

5. De Iongh RU, Wederell E, Lovicu FJ, McAvoy JW. Transforming growth factor-β-

induced epithelial-mesenchymal transition in the lens: a model for cataract formation.

Cells Tissues Organs 2005;179(1-2):43-55.

6. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Investig

2009;119(6):1420.

7. Wells A, Chao YL, Grahovac J, Wu Q, Lauffenburger DA. Epithelial and mesenchymal

phenotypic switchings modulate cell motility in metastasis. Front Biosci 2011;16:815-

837.

8. Takaishi M, Tarutani M, Takeda J, Sano S. Mesenchymal to Epithelial Transition

Induced by Reprogramming Factors Attenuates the Malignancy of Cancer Cells. PloS

one 2016;11(6):e0156904.

This article is protected by copyright. All rights reserved

  

9. Wells A, Yates C, Shepard CR. E-cadherin as an indicator of mesenchymal to epithelial

reverting transitions during the metastatic seeding of disseminated carcinomas. Clin Exp

Metastasis 2008;25(6):621-628.

10. Macıá s•-Silva M, Hoodless PA, Tang SJ, Buchwald M, Wrana JL. Specific activation of

Smad1 signaling pathways by the BMP7 type I receptor, ALK2. J Biol Chem

1998;273(40):25628-25636.

11. Kingsley DM. The TGF-beta superfamily: new members, new receptors, and new genetic

tests of function in different organisms. Genes Dev 1994;8(2):133-146.

12. Kang MH, Kang HN, Kim JL, Kim JS, Oh SC, Yoo YA. Inhibition of PI3 kinase/Akt

pathway is required for BMP2-induced EMT and invasion. Oncol Rep 2009;22(3):525-

534.

13. Zeisberg M, Hanai J-i, Sugimoto H et al. BMP-7 counteracts TGF-β1-induced epithelial-

to-mesenchymal transition and reverses chronic renal injury. Nat Med 2003;9(7):964-

968.

14. Zeisberg M, Kalluri R. The role of epithelial-to-mesenchymal transition in renal fibrosis.

J Mol Med 2004;82(3):175-181.

15. Rahman MS, Akhtar N, Jamil HM, Banik RS, Asaduzzaman SM. TGF-β/BMP signaling

and other molecular events: regulation of osteoblastogenesis and bone formation. Bone

research 2015;3.

16. Chen DI, Zhao M, Mundy GR. Bone morphogenetic proteins. Growth factors

2004;22(4):233-241.

This article is protected by copyright. All rights reserved

  

17. Macıá s-Silva M, Hoodless PA, Tang SJ, Buchwald M, Wrana JL. Specific activation of

Smad1 signaling pathways by the BMP7 type I receptor, ALK2. Journal of Biological

Chemistry 1998;273(40):25628-25636.

18. Hata A, Lagna G, Massagué J, Hemmati-Brivanlou A. Smad6 inhibits BMP/Smad1

signaling by specifically competing with the Smad4 tumor suppressor. Genes &

development 1998;12(2):186-197.

19. Levy L, Hill CS. Alterations in components of the TGF-β superfamily signaling

pathways in human cancer. Cytokine & growth factor reviews 2006;17(1):41-58.

20. Hahn SA, Schutte M, Hoque A et al. DPC4, a candidate tumor suppressor gene at human

chromosome 18q21. 1. SCIENCE-NEW YORK THEN WASHINGTON- 1996:350-352.

21. Muller N, Reinacher-Schick A, Baldus S et al. Smad4 induces the tumor suppressor E-

cadherin and P-cadherin in colon carcinoma cells. Oncogene 2002;21(39):6049-6058.

22. Schwarte-Waldhoff I, Volpert OV, Bouck NP et al. Smad4/DPC4-mediated tumor

suppression through suppression of angiogenesis. Proceedings of the National Academy

of Sciences 2000;97(17):9624-9629.

23. Masetti M, Acquaviva G, Visani M et al. Long-term survivors of pancreatic

adenocarcinoma show low rates of genetic alterations in KRAS, TP53 and SMAD4.

Cancer biomarkers: section A of Disease markers 2017.

24. El-Guendy N, Rangnekar VM. Apoptosis by Par-4 in cancer and neurodegenerative

diseases. Exp Cell Res 2003;283(1):51-66.

25. Cook J, Krishnan S, Ananth S et al. Decreased expression of the pro-apoptotic protein

Par-4 in renal cell carcinoma. Oncogene 1999;18(5):1205-1208.

This article is protected by copyright. All rights reserved

  

26. Kögel D, Reimertz C, Mech P et al. Dlk/ZIP kinase-induced apoptosis in human

medulloblastoma cells: requirement of the mitochondrial apoptosis pathway. Br J Cancer

2001;85(11):1801.

27. Moreno-Bueno G, Fernandez-Marcos PJ, Collado M et al. Inactivation of the candidate

tumor suppressor par-4 in endometrial cancer. Cancer Res 2007;67(5):1927-1934.

28. Zapata-Benavides P, Méndez-Vázquez JL, González-Rocha TR et al. Expression of

prostate apoptosis response (Par-4) is associated with progesterone receptor in breast

cancer. Arch Med Res 2009;40(7):595-599.

29. Goswami A, Burikhanov R, de Thonel A et al. Binding and phosphorylation of par-4 by

akt is essential for cancer cell survival. Mol Cell 2005;20(1):33-44.

30. Shrestha-Bhattarai T, Rangnekar VM. Cancer-selective apoptotic effects of extracellular

and intracellular Par-4. Oncogene 2010;29(27):3873-3880.

31. Rah B, Amin H, Yousuf K et al. A novel MMP-2 inhibitor 3-azidowithaferin A (3-

azidoWA) abrogates cancer cell invasion and angiogenesis by modulating extracellular

Par-4. PloS one 2012;7(9):e44039.

32. Burikhanov R, Sviripa VM, Hebbar N et al. Arylquins target vimentin to trigger Par-4

secretion for tumor cell apoptosis. Nature chemical biology 2014;10(11):924-926.

33. Amin H, Nayak D, Chakraborty S et al. Par-4 dependent modulation of cellular β-catenin

by medicinal plant natural product derivative 3-azido Withaferin A. Mol Carcinog

2016;55(5):864-881.

34. Johnson IT. Glucosinolates: bioavailability and importance to health. Int J Vitam Nutr

Res 2002;72(1):26-31.

This article is protected by copyright. All rights reserved

  

35. Verkerk R, Van der Gaag MS, Dekker M, Jongen WMF. Effects of processing conditions

on glucosinolates in cruciferous vegetables. Cancer Lett 1997;114(1-2):193-194.

36. Chang X, Tou JC, Hong C et al. 3,3'-Diindolylmethane inhibits angiogenesis and the

growth of transplantable human breast carcinoma in athymic mice. Carcinogenesis

2005;26(4):771-778.

37. Nachshon-Kedmi M, Fares FA, Yannai S. Therapeutic activity of 3, 3′-diindolylmethane

on prostate cancer in an in vivo model. Prostate 2004;61(2):153-160.

38. Hong C, Kim H-A, Firestone GL, Bjeldanes LF. 3, 3′-Diindolylmethane (DIM) induces a

G1 cell cycle arrest in human breast cancer cells that is accompanied by Sp1-mediated

activation of p21WAF1/CIP1 expression. Carcinogenesis 2002;23(8):1297-1305.

39. Hong C, Firestone GL, Bjeldanes LF. Bcl-2 family-mediated apoptotic effects of 3, 3′-

diindolylmethane (DIM) in human breast cancer cells. Biochem Pharmacol

2002;63(6):1085-1097.

40. Xue L, Firestone GL, Bjeldanes LF. DIM stimulates IFNγ gene expression in human

breast cancer cells via the specific activation of JNK and p38 pathways. Oncogene

2005;24(14):2343-2353.

41. Kristal AR, Lampe JW. Brassica vegetables and prostate cancer risk: a review of the

epidemiological evidence. Nutr Cancer 2002;42(1):1-9.

42. Saati GE, Archer MC. Inhibition of fatty acid synthase and Sp1 expression by by 3, 3′-

diindolylmethane in human breast cancer cells. Nutr Cancer 2011;63(5):790-794.

43. Azmi AS, Ahmad A, Banerjee S, Rangnekar VM, Mohammad RM, Sarkar FH.

Chemoprevention of pancreatic cancer: characterization of Par-4 and its modulation by

3,3′ diindolylmethane (DIM). Pharm Res 2008;25(9):2117-2124.

This article is protected by copyright. All rights reserved

  

44. Sharma DK, Rah B, Lambu MR et al. Design and synthesis of novel N, N′-glycoside

derivatives of 3, 3′-diindolylmethanes as potential antiproliferative agents.

Medchemcomm 2012;3(9):1082-1091.

45. Nayak D, Amin H, Rah B et al. A therapeutically relevant, 3,3'-diindolylmethane

derivative NGD16 attenuates angiogenesis by targeting glucose regulated protein, 78kDa

(GRP78). Chem Biol Interact 2015;232:58-67.

46. Torres MP, Rachagani S, Souchek JJ, Mallya K, Johansson SL, Batra SK. Novel

pancreatic cancer cell lines derived from genetically engineered mouse models of

spontaneous pancreatic adenocarcinoma: applications in diagnosis and therapy. PloS one

2013;8(11):e80580.

47. Martin KH, Hayes KE, Walk EL, Ammer AG, Markwell SM, Weed SA. Quantitative

measurement of invadopodia-mediated extracellular matrix proteolysis in single and

multicellular contexts. J Vis Exp 2012(66).

48. Ghosh S, Basu M, Roy SS. ETS-1 protein regulates vascular endothelial growth factor-

induced matrix metalloproteinase-9 and matrix metalloproteinase-13 expression in human

ovarian carcinoma cell line SKOV-3. J Biol Chem 2012;287(18):15001-15015.

49. Wellner U, Schubert Jr, Burk UC et al. The EMT-activator ZEB1 promotes

tumorigenicity by repressing stemness-inhibiting microRNAs. Nature cell biology

2009;11(12):1487.

50. Hahn SA, Schutte M, Hoque A et al. DPC4, a candidate tumor suppressor gene at human

chromosome 18q21. 1. Science 1996;271(5247):350-353.

51. Miyaki M, Iijima T, Konishi M et al. Higher frequency of Smad4 gene mutation in

human colorectal cancer with distant metastasis. Oncogene 1999;18(20).

This article is protected by copyright. All rights reserved

  

52. Muller N, Reinacher-Schick A, Baldus S et al. Smad4 induces the tumor suppressor E-

cadherin and P-cadherin in colon carcinoma cells. Oncogene 2002;21(39):6049-6058.

53. Xia Y, Shen S, Verma IM. NF-kB, an active player in human cancers. Cancer

immunology research 2014;2(9):823-830.

54. Hoesel B, Schmid JA. The complexity of NF-kB signaling in inflammation and cancer.

Molecular cancer 2013;12(1):86.

55. Buijs JT, Henriquez NV, Van Overveld PGM et al. Bone morphogenetic protein 7 in the

development and treatment of bone metastases from breast cancer. Cancer Res

2007;67(18):8742-8751.

56. Buijs JT, Rentsch CA, van der Horst G et al. BMP7, a putative regulator of epithelial

homeostasis in the human prostate, is a potent inhibitor of prostate cancer bone metastasis

in vivo. Am J Pathol 2007;171(3):1047-1057.

57. Na Y-R, Seok S-H, Kim D-J et al. Bone morphogenetic protein 7 induces mesenchymal-

to-epithelial transition in melanoma cells, leading to inhibition of metastasis. Cancer Sci

2009;100(11):2218-2225.

58. Notting I, Buijs J, Mintardjo R et al. Bone morphogenetic protein 7 inhibits tumor growth

of human uveal melanoma in vivo. Invest Ophthalmol Vis Sci 2007;48(11):4882-4889.

59. Bi W-R, Jin C-X, Xu G-T, Yang C-Q. Bone morphogenetic protein-7 regulates Snail

signaling in carbon tetrachloride-induced fibrosis in the rat liver. Exp Ther Med

2012;4(6):1022-1026.

60. Zhou XD, Agazie YM. Inhibition of SHP2 leads to mesenchymal to epithelial transition

in breast cancer cells. Cell Death Differ 2008;15(6):988-996.

This article is protected by copyright. All rights reserved

  

61. Farmakovskaya M, Khromova N, Rybko V, Dugina V, Kopnin B, Kopnin P. E-Cadherin

repression increases amount of cancer stem cells in human A549 lung adenocarcinoma

and stimulates tumor growth. Cell cycle 2016;15(8):1084-1092.

62. Qin Q, Xu Y, He T, Qin C, Xu J. Normal and disease-related biological functions of

Twist1 and underlying molecular mechanisms. Cancer Res 2012;22(1):90-106.

63. Vesuna F, van Diest P, Chen JH, Raman V. Twist is a transcriptional repressor of E-

cadherin gene expression in breast cancer. Biochem Biophys Res Commun

2008;367(2):235-241.

This article is protected by copyright. All rights reserved

  

Figure legends

Figure 1. Reversal of EMT and induction of MET by NGD16. (A) The MET induction by

NGD16 was assessed by treating Panc-1 and MIA Paca-2 cells with indicated concentrations of

NGD16 for 48 h followed with western blotting to check the expression of E-cadherin, ALK2,

Vimentin, Twist-1 and Sox2. (B) Panc-1 and MIA Paca-2 cells were incubated with 2 and 3 µM

of NGD16, respectively, for 0, 12, 24, 36 and 48 h. Whole cell lysates were subjected to Western

blotting for E-cadherin, ALK2, Vimentin, Twist-1 and Sox2. Beta-actin was used as a loading

control. (C) Immunocytochemistry analysis of NGD16-treated Panc-1 cells for E-cadherin

(green, 20x magnification), Vimentin (red, 60x magnification), nuclear DAPI (blue). Scale bar

(20 µm). (D) Panc-1 and MIA Paca-2 cells were treated with indicated concentrations of NGD16

for 48 h and the matrigel invasion assay was carried out. The number of invaded cells in each

treatment were indiscriminately counted from five fields and photographed under an inverted

microscope (20x magnification).(E) Corresponding bar-graph for matrigel invasion assay

representing the number of invading cells per field (F) The anti-metastatic effect of NGD16 was

assessed by culturing Panc-1 cells on FITC-conjugated gelatin matrix with the indicated

conditions. TGF-β1 treated Panc-1 cells were used as a positive control for gelatin degradation.

The threshold areas of degradation were processed and analyzed through Image-J software. (G)

Corresponding bar-graph for FITC-gelatin degradation assay representing the percent area of

degradation. All experiments were performed at least thrice prior to the statistical analysis. Data

were compared with untreated control. S.D. of three independent experiments. *P<0.05,

**P<0.01.

This article is protected by copyright. All rights reserved

  

Figure 2. NGD16 modulates the ALK2/SMAD4 signalling axis to induce MET. (A) NGD16-

treated Panc-1 and MIA Paca-2 cells with the indicated concentrations were subjected to western

blotting for E-cadherin, ALK2 and Smad4. Beta-actin was used as a loading control. (B) BMP7-

treated Panc-1 cells were analyzed for E-cadherin, ALK2 and Smad4 protein levels in a dose-

responsive manner.(C) Immunocytochemistry analysis of Panc-1 cells treated with 2 µM NGD16

and immunostained with ALK2 (red, 60x magnification) along with nuclear DAPI (blue) was

carried out (Scale bar 20 µm) (D) HCT-116 (Smad4+/+), HT-29 (Smad4+/-) and BxPC-3

(Smad4+/-) cells were treated with the above mentioned concentrations of NGD16 and analysed

for ALK2 and E-cadherin expression through immunoblotting. (E) Panc-1 Cells were treated

with TGF-β1 (5 ng/mL) for 48 h in the presence or absence of indicated NGD16 concentrations

and induction of MET phenotype was assessed by observing the cell morphological changes.

The data is representative of three independent experiments performed.

Figure 3. NGD16-mediated E-cadherin upregulation is Smad4-dependent. (A) To confirm

the Smad4-mediated E-cadherin upregulation, MIA Paca-2 cells were transfected with scrambled

siRNA/siRNA Smad4 followed by treatment with NGD16 for 48 h and were subjected to

western blotting for the expression of Smad4 and E-cadherin. Beta-Actin was used as a loading

control. (B) The similar knockdown conditions were employed in Panc-1 cells and the cells

were then subjected to immunocytochemistry analysis for Smad4 (40x magnification) and E-

cadherin (20x magnification) expression. Scale bar, 20 µm (C) HCT-116 (Smad4+/+) and HT-29

(Smad4+/-) cells were treated with Vehicle as well as the indicated concentrations of NGD16 for

48 h and immunostained to visualize the E-cadherin (green) localization and DAPI (blue).

Photographs were taken at 20x magnification. Results were compared with the untreated control

This article is protected by copyright. All rights reserved

  

and images were taken from at least three random fields and. The data is representative of three

independent experiments performed. Scale bar, 20 µm.

Figure 4. NGD16 induces the endogenous Par-4 levels and negatively regulates EMT. (A)

To assess the NGD16-induced endogenous Par-4 levels, Panc-1 and MIA Paca-2 cells were

treated with varying concentrations of NGD16 as indicated. The whole cell lysates were

examined for the expression of Par-4 through immunoblotting. Beta-actin was used as a loading

control. (B) Time-dependent upregulation of Par-4 was evaluated in Panc-1 and MIA Paca-2

cells upon NGD16 treatment for the mentioned time intervals. (C) Immunochemistry analysis

was also carried for visualization of the NGD16-induced Par-4 (green) and DAPI (blue) levels in

Panc-1 and MIA Paca-2 upon treatment with vehicle or the indicated NGD16 concentrations for

48 h. Images were captured at 20x magnification. (D) Panc-1 and MIA Paca-2 cells were

transiently transfected with GFP/GFP-Par-4 and analyzed by western blot analysis for Par-4, E-

cadherin and Vimentin expression along with beta-actin. (E) The bar-graph represents the

intensity of protein expression quantified by densiometric analysis. (F) Immunocytochemistry of

the Panc-1 cells transiently transfected with GFP/GFP-Par-4 (green) for 24 h was carried out to

analyze the Vimentin (red) expression along with DAPI (blue) . Photographs were taken at 65x

magnification. Scale bar 20 µm. (G) To examine the anti-invasive effect of Par-4, Panc-1 cells

were transfected with GFP/GFP-Par-4 and subjected to the matrigel invasion assay. Invaded cells

in each treatment were randomly counted from five fields and photographed under an inverted

microscope (20x magnification). (H) The Bar-graph represents the number of invading cells per

field. *P < 0.05, **P < 0.01.

Figure 5. NGD16-induced Smad4 induction and the consequent E-cadherin upregulation is

Par-4-dependent. (A) MIA Paca-2 and Panc-1 cells were treated with increasing concentrations

This article is protected by copyright. All rights reserved

  

of NGD16, as indicated. The whole cell lysates were analyzed for the endogenous Par-4, E-

cadherin, Vimentin, Twist-1 and NF-kB (p65) levels along with beta-actin. (B) MIA Paca-2 and

Panc-1 cells transiently transfected with GFP/GFP-Par-4 were subjected to immunoblotting to

check the ALK2, Smad4 and beta-actin levels. (C) The bar-graph represents the intensity of

protein expression as quantified by the densitometric analysis. (D) Panc-1 cells were transfected

with scramble (control) siRNA/siRNA Par-4 as indicated and then followed with indicated

vehicle/NGD16 treatment. The cell lysates from above were subjected to Western blot analysis

for determining the expression of Par-4, ALK2, Smad4, NF-kB (p65) and Vimentin along with

beta-actin.(E) The similar conditions were replicated in Panc-1 cells for immunocytochemistry

analysis of E-cadherin (green) expression and localization. The images were taken at 20x

magnification. Scale bar, 20 µm. (F) Twist-1 transcription activity was assessed using a Twist-1

reporter construct (PGL3-Twist-1-luc) and a luciferase reporter assay system in the presence of

Vehicle/NGD16/GFP/GFP-Par-4 as indicated. The fold increase was calculated compared to the

untreated control. Data are presented as means ± S.D. from at least three independent

experiments. *P < 0.05, **P < 0.01.

Figure 6. NGD16 decreases the metastatic incidence in syngenic mouse metastatic

pancreatic cancer model. (A) The gross necropsy analysis of the given organs depicting visible

metastatic tumor nodules in the Vehicle as well as NGD16-treated group. (B) The Bar-graph

represents the percent metastasis incidence in the vehicle/ NGD16-treated group as analysed by

the gross necropsy analysis. (C) Incidence of ascites in the Vehicle/ NGD16-treated group are

represented. (D) The graph shows the overall percent survival of the animals from the vehicle

treated group as well as the NGD16 treated group. (E) Tumor tissue lysates obtained from

NGD16 injected mice and control mice were probed to check the expression of Par-4. Beta-actin

This article is protected by copyright. All rights reserved

  

was used as a loading control. Data are presented as means ± S.D. from at least three

independent experiments. *P < 0.05, **P < 0.01

Figure 7. Schematic diagram depicts the proposed mechanism of Par-4 action in MET

induction and metastasis prevention. Treatment with NGD16 results in Par-4 induction that

activates MET by upregulating ALK2 and Smad4, which along with Smad1/5/8 translocates to

the nucleus and results in E-cadherin upregulation. Par-4 also impedes the Twist-1

transcriptional activity that may also lead to increased E-cadherin protein levels.

This article is protected by copyright. All rights reserved

  

Figure 1

This article is protected by copyright. All rights reserved

  

Figure 2

This article is protected by copyright. All rights reserved

  

Figure 3

This article is protected by copyright. All rights reserved

  

Figure 4

This article is protected by copyright. All rights reserved

  

Figure 5

This article is protected by copyright. All rights reserved

  

Figure 6

This article is protected by copyright. All rights reserved

  

Figure 7

This article is protected by copyright. All rights reserved