Cyclodipeptide c(Orn-Pro) Conjugate with 4-Ethylpiperic Acid Abrogates Cancer Cell Metastasis through Modulating MDM2

Article abstract of DOI:10.1021/acs.bioconjchem.7b00670

The present work describes the synthesis, characterization, and anticancer properties of c(Lys-Pro), P1; c(Orn-Pro), P2; and conjugates PA-c(Lys-Pro), C1; PA-c(Orn-Pro), C2; EPA-c(Lys-Pro), C3; and EPA-c(Orn-Pro), C4. Among all, conjugate C4 displays pote

Full text of DOI:10.1021/acs.bioconjchem.7b00670

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Article
Cyclodipeptide c(Orn-Pro) Conjugate with 4-Ethylpiperic Acid (EPA)
Abrogates Cancer Cells Metastasis Through Modulating MDM2
Sudha Shankar, Mir Mohd Faheem, Debasis Nayak, Naiem Ahmad Wani,
Saleem Farooq, Surrinder Koul, ANINDYA GOSWAMI, and Rajkishor Rai
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00670 • Publication Date (Web): 07 Dec 2017
Downloaded from http://pubs.acs.org on December 8, 2017
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Cyclodipeptide c(Orn-Pro) Conjugate with 4-Ethylpiperic Acid (EPA) Abrogates
Cancer Cells Metastasis Through ModulatingMDM2
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Sudha Shankar,ad#Mir Mohd Faheem,b# Debasis Nayak,bd Naiem Ahmad Wani,a Saleem Farooq,c
Surrinder Koul,c Anindya Goswami,bd* Rajkishor Rai,ad*
aMedicinal Chemistry Division, CSIRꢀIndian Institute of Integrative Medicine, Canal Road, Jammuꢀ
180001, India.
bCancer Pharmacology Division, CSIRꢀIndian Institute of Integrative Medicine, Canal Road,
Jammuꢀ180001, India.
cBioorganic Chemistry Division, CSIRꢀIndian Institute of Integrative Medicine, Canal Road,
Jammuꢀ180001, India.
d Academy of Scientific and Innovative Research, New Delhi, India
# Contributed equally
Corresponding Author
*A.G.: Phoneꢀ +91ꢀ191ꢀ 2585006 ext. 224; Eꢀmail: agoswami@iiim.ac.in.
*R. R.: Phoneꢀ +91ꢀ191ꢀ2585006 ext. 268; Eꢀmail: raj@iiim.ac.in
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Abstract
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The present work describes the synthesis, characterization and anticancer properties of c(LysꢀPro),
P1; c(OrnꢀPro), P2 and conjugates PAꢀc(LysꢀPro), C1; PAꢀc(OrnꢀPro),C2; EPAꢀc(LysꢀPro), C3 and
EPAꢀc(OrnꢀPro), C4.Among all,conjugateC4 displays potent antiꢀcancer activity with IC50 1.3ꢀM in
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MDAꢀMBꢀ231, 3.5 µM in PCꢀ3, 8.9ꢀM in MCFꢀ7 and 9.6 ꢀM in Miapacaꢀ2 cancer cells.In
addition,C4downregulates the expression of MDM2 and abrogates the cancer cell
invasion/metastasis. Through knockꢀdown of MDM2, we demonstrate that this abrogation of
metastasis by C4 is primarily MDM2 dependent. Further, the animal studies underscorethe
antitumor as well as antimetastatic potential of C4in vivo in breast cancer model at a safe and
tolerable dose of 20 mg/kg.
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Introduction
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Cyclodipeptides i.e. 2,5ꢀdiketopiperazines are well described in diverse natural products
including fungi, bacteria and the plant kingdom having varied biological activities viz. antiꢀ
cancer,1 quorum sensing,2 and antibacterial.3 The cyclodipeptides scaffolds have been
substantially investigated in the search for new class of MDM2/p53 inhibitors.4 The essential
molecule that could perturb the MDM2/p53 interaction is being considered as a conscientious
topic in the field of emerging antiꢀmetastasistherapeutics.5 MDM2 possesses key functional
regulation of p53 as principal cellular antagonist of p53 by limiting the p53 tumor suppressor
function through its degradation.6,7 Intriguingly, MDM2 is rising up as a key player in
synchronizing invasiveness and metastasis. Several authentic reports have demonstrated that
amplification of the MDM2 gene correlates with the metastatic stage and in turn with the
prognosis of the disease.8 The over expression of MDM2 is deeply corroborated with tumors
that have a higher degree of migration, invasiveness, greater metastatic potentiality and
resistance to chemotherapeutic agents and radiation.9, 10
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Metastasis, the ability of cancer cells to relocate from their primary niche to distant sites,
is the most fearsome aspect of cancer. Statistics reveal that local invasion and metastasis to
distant organs account for a significant proportion of cancerꢀrelated deaths worldwide.11
Relying on conventional treatment strategies is unlikely to produce any significant improvements
in therapy as they fail to counter the severity of metastasis. Cellular stresses such as DNA
damage, hypoxia, telomere shortening, and oncogene activation are despondent factors for
almost all the cancers leading to the stressꢀmediated stabilization of tumor suppressor p53.12
Primarily, Mouse Double Minute Homologue 2ꢀMDM2 (an oncogene) is accountable for the
regulation of p53 and is the principal cellular antagonist of p53 by limiting the p53 tumor
suppressor function through its degradation. MDM2 triggers E3 ubiquitin ligase activity
investigating the proteasomal degradation of p53 by catalyzing its ubiquitination; but in turn,
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MDM2 is transactivated by p53.13 Gene amplification and protein over expression of MDM2 are
often associated with certain types of cancers indicating that MDM2 plays a pivotal role in
tumorigenesis. It confers to tumor cells the ability to bypass apoptosis, facilitating tumor growth
and progression.6, 14 MDM2 is expressed at high levels in numerous human cancers, including
those of the breast, lung, colon, pancreas, and hematological malignancies lymphomas and
leukemias.15 Although the mechanism by which MDM2 contributes to tumor initiation has been
validated with the identification of multiple interacting proteins, the other vital
physiological/pathological roles of MDM2 remain obscure.16,17 Nonꢀcanonically, the role of
MDM2 as a key player in invasiveness and metastasis is an emerging field. Interestingly, quite a
few recent reports have demonstrated that amplification of the MDM2 gene correlates with the
metastatic stage and in turn with the prognosis of the disease.18 Moreover, several studies have
also shown that MDM2 over expression is associated with tumors that have a higher degree of
invasiveness and resistance to chemotherapeutic agents and radiation.8, 15
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Herein, we synthesized the cyclic dipeptides c(OrnꢀPro), P1; c(LysꢀPro), P2 and their
conjugates with piperic acid (PA) and 4ꢀethylpiperic acid (EPA), PAꢀc(LysꢀPro), C1; PAꢀc(Ornꢀ
Pro), C2; EPAꢀc(LysꢀPro), C3 and EPAꢀc(OrnꢀPro),C4. The conjugates C1-C4 were synthesized
based on the novel strategy to conjuate the diketopiperazine scaffold derived from 52RR with
piperic/4ꢀethylpieric acid (Figure 1) in order to selectively explore the p53ꢀMDM2 interaction.
52RR has been developed by the side chain modification of the 2,5ꢀDKP’s scaffold and
exhibited the inhibition of MDM2ꢀp53 interaction with IC50 31ꢁM.4 The chemical structure of
PA, EPA, cyclic peptides and conjugates of PA and EPA is shown in Figure 2. Piperic acid was
obtained from the hydrolysis of piperine, an alkaloid present in black pepper (Piper nigrum-L/
Piper longum).19 Piperine has been reported to suppress the tumor growth and metastasis in 4T1
murine breast cancer model.20 It also induces apoptotic cell death in human melanoma, lung and
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colorectal carcinoma cell lines, and inhibits the proliferation of human prostate cancer cells via
induction of cellꢀcycle arrest and autophagy at G0/G1 phase.21
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Figure 1. Structure of compound 52RR and conjugates C1ꢀC4
Figure 2.Chemical structure of piperic acid, 4ꢀethylpiperic acid, cyclic dipeptides c(OrnꢀPro),
P1; c(LysꢀPro), P2 and conjugates PAꢀc(LysꢀPro), C1; PAꢀc(OrnꢀPro), C2; EPAꢀc(LysꢀPro), C3
and EPAꢀc(OrnꢀPro),C4.
Among all the synthesized conjugates, C4 exhibited promising cytotoxic activity against
multiple cancer cell lines of various origins. C4 evolved as most potent conjugate in terms of
cytotoxicity and exhibited IC50 values: 1.3ꢀM in MDAꢀMBꢀ231; 3.5µM in PCꢀ3, 8.9ꢀM in
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MCFꢀ7 and 9.6ꢀM in Miapacaꢀ2 cells. Further, the conjugate C4 was evaluated as down
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regulator of MDM2 expression and its role in abrogating invasion/metastasis.
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Results and Discussion
Chemistry
Piperic acid (PA) was obtained from the hydrolysis of piperine, an alkaloid, present in black
pepper. Piperine has been used as bioenhancer.22, 23 4ꢀEthylpiperic acid (EPA) was synthesized
by the procedure reported in literature.24 The cyclic dipeptides, c(LysꢀPro), P1 and c(OrnꢀPro),
P2 were synthesized in solution phase by coupling of BocꢀLys(Cbz)ꢀOH/ BocꢀOrn(Cbz)ꢀOH
with LꢀProꢀOMe.HCl using 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide hydrochloride
(EDC.HCl) and Nꢀmethylmorpholine (NMM) in dry dichlomethane followed by cyclization after
Boc deprotection in presence of dry N,Nꢀdimethylformamide (DMF) and piperidine as reported
in the literature.2 The removal of carboxybenzyl (Cbz) was performed using H2/Pd in methanol
(Scheme 1).
Scheme1. Synthesis of cyclic dipeptidesP1 and P2
Reagents and Conditions: a) BocꢀLys(z)/Orn(z), dry DCM, NMM, EDC.HCl, LꢀProꢀOMe.HCl,
12h; b) 30%TFA in DCM, 3h, rt; c) piperidine, DMF, Reflux; d) 10% Pd/C, H2, MeOH.
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Further, the conjugates C1-C4 were preapred by coupling of cyclic dipeptides c(OrnꢀPro), P1
and c(LysꢀPro), P2 with piperic acid and 4ꢀethylpiperic acid using EDC.HCl/1ꢀ
hydroxybenzotriazole (HOBt) in dry DMF and Nꢀmethylmorpholine (Scheme 2).
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Scheme2. Synthetic scheme of conjugates C1ꢀC4
Reagents and conditions: a) Piperic acid (PA),Dry CH2Cl2, NMM, EDCI, [c(LysꢀPro)/c(Ornꢀ
Pro)], 0°C to room temp; b) 4ꢀEthylpiperic acid (EPA), Dry CH2Cl2, NMM, EDCI, [c(Lysꢀ
Pro)/c(OrnꢀPro)], 0°C to room temp.
Biology
C4 is a potent cytotoxic agent
Diketopiperzines were earlier reported as antagonists of proteinꢀprotein interaction25 but
diketopiperazine conjugates as cytotoxic agents are not reported so far and detail biological
activity of diketopiperazine conjugates is yet to be explored. Therefore, the synthesis of new
conjugates containing the diketopiperazine skeleton backbone was carried out. In order to
evaluate the cytotoxic potential of the peptides and conjugates synthesized, MTT assay was
performed. Among all the conjugates, C4 was found to be the most potent molecule against
various cancer cell lines in the series with IC50 values of 1.3µM, 3.5µM, 8.9µM 9.6µM, and
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31µM in MDAꢀMBꢀ231, PCꢀ3, MCFꢀ7, MiaPacaꢀ2, and HCTꢀ116, respectively (Table 1).
Importantly, against normal breast epithelial cell line fRꢀ2, C4 showed minimum toxicity (IC50
value of 74.3 µM), indicating a higher therapeutic index. Further, upon diligent analysis of the
screening data, it was evident that C4 showed better cytotoxic potential against aggressive
mesenchymal cells (MDAꢀMBꢀ231, PCꢀ3 and MiaPacaꢀ2) in comparison to the epithelial cells
(MCFꢀ7 and HCTꢀ116). Accordingly, the MDAꢀMBꢀ231 and PCꢀ3 cell lines depicting potent
IC50 values were employed for further mechanistic studies.
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Table 1. IC50 values (µM) for the compounds screened against various cell lines.
Compound
PA
MDAꢀMBꢀ231 MCFꢀ7
HCTꢀ116
>100
MiaPacaꢀ2
>100
PCꢀ3
>100
fRꢀ2
ꢀꢀ
>100
>100
>100
>100
EPA
P1
>100
>100
>100
ꢀꢀ
>100
>100
>100
>100
>100
ꢀꢀ
P2
>100
>100
>100
>100
>100
ꢀꢀ
C1
>100
>100
>100
24.3±0.90
7.9±0.40
8.5±0.72
9.6± 0.13
43±0.90
>100
C2
43±1.9
13±0.89
1.3±0.29
75± 3.4
23±0.91
8.9±0.76
>100
5.1±0.27 89.9±1.6
29±0.95 ꢀꢀ
3.5±0.48 74.3±1.8
C3
49±1.4
31±1.65
C4
C4 inhibits MDM2 irrespective of the p53 status of the cell
Structural studies have revealed that diketopiperazine derivatives could be considered as
potential obstructer of p53ꢀMDM2 interaction (data not shown). To check the effect of C4 on
MDM2 expression in MDAꢀMBꢀ231 and PCꢀ3 cells, concentration and time dependent studies
were carried out by western blotting and immunocytochemistry. Our western blot results showed
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a steady decline of MDM2 in a dose dependent manner. In both cell lines, a significant
reduction in MDM2 expression was achieved at 1, 2 and 3µM concentrations of C4, whereas in
HCTꢀ116 and MCFꢀ7 cells, significant downregulation of MDM2 was also observed at 2 and
3µM concentration of C4 (Figure 3A).
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Figure 3. C4 downregulates the expression of MDM2 in MDAꢀMBꢀ231 and PCꢀ3 cells. (A)
Dose dependent inhibition of MDM2 by C4 in MDAꢀMBꢀ231, PCꢀ3, MCFꢀ7, HCTꢀ116 and
Miapacaꢀ2 cells at 24h by western blotting. (B) Time dependent inhibition of MDM2 by C4
(3µM) in MDAꢀMBꢀ231 and PCꢀ3 cells by western blotting. βꢀActin expression was considered
to ensure equal loading. (C) Immunocytochemistry images depicting the down modulation of
MDM2 by C4 (3µM) at 24h in MDAꢀMBꢀ231 and PCꢀ3 cells. The data represents three
independent experiments performed separately.
However, negligible reduction in the expression of MDM2 was noticed in Miapacaꢀ2 cells.
Further, by evaluating the time dependent effects of C4, a drastic attenuation in MDM2
expression was observed at 3µM concentration beyond 24 hours (Figure 3B). Accordingly, a
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dose of 3µM was selected for further studies. The molecular dynamics between MDM2 and p53
lying within a cell are tightly regulated through a feedback loop that mutually exists between
these two proteins. It is pertinent to mention here that C4 was unable to enhance the p53
expression, even though it robustly alleviated the MDM2 expression in the cell lines tested (data
not shown). Interestingly, our results clearly demonstrated that the inhibition of MDM2 by C4
was independent of the p53 status of the cell. This was evident through the downregulation of
MDM2 in p53ꢀwild type (HCTꢀ116 and MCFꢀ7), p53ꢀmutant (MDAꢀMBꢀ231 and MiaPacaꢀ2)
and p53ꢀnull (PCꢀ3) cells. Additionally, the immunocytochemistry results (Figure 3C) were in
accordance with the western blots results showing a marked decrease in MDM2 expression at
3µM (24 h) concentration of C4 in both the cell lines tested. Further, upon careful examination
of the morphology of the cells, it was observed that C4 triggered proꢀepithelial phenotype at
3µM concentration tempting possible indications towards the antiꢀmetastatic potential of C4.
These results collectively demonstrate that C4 inhibits expression of MDM2 in multiple cancer
cells.
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C4 abrogates the metastatic character of MDA-MB-231 and PC-3 cells
In order to check the effects of C4 on key proteins facilitating the EMT phenomenon of cancer
cells, highly aggressive/invasive cell lines MDAꢀMBꢀ231 and PCꢀ3 were selected for metastasis
related experiments. The expression levels of Eꢀcadherin (a protein more associated with the
epithelial character of a particular cell) were steadily elevated in a dose dependent manner upon
treatment with C4 (Figure 4A). Sequestering/ubiquitnylation of Eꢀcadherin in the endosome have
been reported to facilitate the metastatatic phenotype in cancer cells.26 Immunoblot analysis with
Snail, which is a negative repressor of Eꢀcadherin showed reciprocal effect of C4 on Snail in
both the cell lines tested at the aforementioned dose and time point. Twistꢀ1 is another important
protein involved in EMT and is upstream of Snail. It binds to the promoter of Snail, thereby,
acting as its coꢀtranscriptional activator and drives the expression of Snail.27 Of note, C4 also
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resulted in abrogating Twistꢀ1 in a dose dependent manner. Moreover, our results clearly
exhibited a clear attenuation in the expression of Vimentin, a cytoskeleton protein which
modulates the epithelial phenotype of the cell. Matrixmetalloproteases (MMPs) are a class of
metalloproteases involved in the remodeling of the extracellular matrix (ECM).28 MMP9 and
MMP2 are representative candidates of this class.
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Figure 4. C4 abrogates the migration and invasion capabilities of MDAꢀMBꢀ231 and PCꢀ3 cells.
(A) C4 modulates the expression of EMT markers in MDAꢀMBꢀ231 and PCꢀ3 cells. The cells
were treated with 1, 2 and 3µM of C4 or vehicle and checked for the protein expression of Eꢀ
cadherin, Vimentin, Twistꢀ1, Snail, MMP2, MMP9 and TIMPꢀ1 through western blotting. βꢀ
Actin was used as loading control. (B) Gelatin zymography profiles of MMP2 and MMP9 in
MDAꢀMBꢀ231 and PCꢀ3 cells. Bovine serum albumin was used as a loading control. (C)
Wound healing assay. MDAꢀMBꢀ231 and PCꢀ3 cells were treated with indicated concentrations
of C4 or vehicle for 24 h after giving the wound to the confluent monolayer of cells. The
scratched areas were photographed (20x) at zero hour and at 24 h to assess the degree of wound
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healing. (D) Cells were treated with indicated concentrations of C4 for 24 h and the matrigel
invasion (Boyden) assay was performed. Migrating cells located in the bottom insert were
photographed under an inverted microscope (10 x magnifications). (E) MDAꢀMBꢀ231 and PCꢀ3
cells were treated with VEGF alone or in combination with 3µM of C4 for 24 h. Cells scattered
from the individual colonies were observed under an inverted microscope and photographed
(10x).
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ECM remodeling is one of the first steps involved in the metastatic phenomena. To corroborate
the effect of C4 on ECM degradation, both western blot analysis and gelatin zymography
profiles (Figure 4B) of MMP9 and MMP2 were studied. It was observed that C4 treatment
resulted in the inhibition of both MMP9 and MMP2 expression as well as their gelatinase
activity is evident by their western blots and zymography profiles. Besides, C4 also drastically
augmented TIMPꢀ1expression, the pan natural inhibitor of MMPs, in both MDAꢀMBꢀ231 and
PCꢀ3 cells. Further, we also determined the effect of C4 on migration of cancer cells by scratch
motility assay. Our results implied that C4 abrogated the migration of MDAꢀMBꢀ231 and PCꢀ3
cells (Figure 4C). At 3µM dose of C4, the number of migratory cells was down by 63% in
MDAꢀMBꢀ231 cells.Similar results were also observed in PCꢀ3 cells. A significant reduction of
55% in the number of migratory cells was observed at the indicated dose (Supporting Figure
S1A). Besides ECM remodeling, migration and invasion are other key hallmarks of metastatic
cancer cells. The effect of C4 on migration was ascertained by the Matrigel Invasion assay. This
assay is used to evaluate the invasion/migration potential of cancer cells through their ability to
invade through biological matrices in vitro. Of note, following treatment with the indicated doses
of C4 (Figure 4D), lesser cells were able to invade through the membrane. In MDAꢀMBꢀ231
cells, in comparison to vehicle, at 1µM dose, only 34% cells were able to migrate through the
upper insert of the chamber, while as at 3µM dose, only 16% cells were able to invade the matrix
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(Supporting Figure S1B). Similarly, in PCꢀ3 cells in comparison to vehicle, only 29% and 8%
cells were able to migrate and invade at 1 and 3µM concentrations of C4, respectively. Cell
scattering is the predominant feature of mesenchymal cancer cells; MDAꢀMBꢀ231 and PCꢀ3
cells were stimulated with VEGF (chemical enhancer of cell motility) alone or in the presence of
3µM concentration of C4 diluted in growth medium (Figure 4E). Scattered colonies were found
in both these cell lines when treated with VEGF alone. However, the extent of scattering was
more profound in MDAꢀ MBꢀ231 cells as compared to PCꢀ3 cells. This can be attributed to the
highly invasive character of MDAꢀMBꢀ231 cells. Upon treatment with 3µM dose of C4, the
scattering phenotype was reversed significantly. The reversal in scattering was modest and
noteworthy in PCꢀ3 cells characterized by a change in morphology due to the cellꢀcell reꢀ
association. In VEGF alone treatment or in vehicle treated cells, the acquisition of a migratory,
fibroblastꢀlike phenotype was sharply distinguishable whereas that phenotype was revoked in C4
treated cells. Further, to check the effect of C4 on the matrix degradation, we employed the
fluorescent gelatin matrix degradation assay. The ability of MDAꢀMBꢀ231 and PCꢀ3 cells to
degrade the matrix was examined by culturing the cells on a crosslinked fluorophore (FITC)ꢀ
conjugatedꢀgelatin matrixꢀcoated coverslips for 24 h. The results clearly depicted that higher
concentration (3µM) of C4 abolished the matrix gelatin degradation by these aggressive cell
lines. In comparison to the vehicle, in MDAꢀMBꢀ231, the percent threshold area of gelatin
degradation was reduced by 50.5%and 87% at 1µM and 3µM concentration of C4, respectively
(Supporting Figure S1C). For PCꢀ3 cells, the percent threshold area of gelatin degradation was
reduced by 66% and 90% at 1µM and 3 µM of C4, respectively. Migration and invasion are key
hallmarks of metastatic cells and C4 significantly attenuated both the migration and invasion
capabilities of MDAꢀMBꢀ231 cells. These results collectively indicate the antiꢀmetastatic
potential of C4.
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MDM2 modulation by C4 is a key to its antimetastatic potential
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To assess whether the antimetastatic potential of C4 was due to its inhibition of MDM2, MDAꢀ
MBꢀ231 and PCꢀ3 cells were transfected with MDM2 siRNA and corresponding nonspecific
scramble control. A significant knockdown of MDM2 was obtained (Figure 5A).
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Figure 5. MDM2 is essential for abrogation of metastasis by C4. (A) Whole cell lysates of
MDAꢀMBꢀ231 and PCꢀ3 cells were subjected to western blot analysis to assess the effect of C4
and siRNA MDM2 on Eꢀcadherin and relevant EMT markers viz. Vimentin, Snail and Twistꢀ1.
βꢀActin was used as a loading control. (B) Western blot analysis of whole cell lysates of MDAꢀ
MBꢀ231 and PCꢀ3 cells transfected with pCMV/ pCMV MDM2 and/ or treated with C4. MDM2,
Eꢀcadherin, Vimentin, Twistꢀ1 and Snail blots are given. (C) Immunocytochemistry of PCꢀ3
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cells treated or transfected with indicated conditions and then stained for MDM2 (green), nuclear
DAPI (blue) and merge. Scale bar, 100µM. (D) Gelatin matrix degradation assay. The
antimetastatic effect of C4 was assessed by culturing PCꢀ3 cells on FITC conjugated gelatin
matrix. ImageꢀJ software was used to process and analyze the threshold areas of degradation.
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Immunoblot analysis of Eꢀcadherin and relevant mesenchymal markers were explored to
ascertain whether the knockdown of MDM2 and exposure to C4 impersonate each other in
terms of their respective modulation of EMT markers. Our results showed that both the MDM2
siRNA and C4 stimulated the expression of Eꢀcadherin in a comparable manner. Further, both
these conditions (knock down of MDM2 as well as C4 treatment) also contributed in the down
modulation of key EMT markers viz., Snail, Twistꢀ1 and Vimentin, in a comparable fashion. To
rule out the offꢀtarget effects of MDM2 siRNA, we carried out the above experiment with two
different sets of siRNA from different regions having insignificant effects on MDM2 protein
levels and the results implied steady downregulation of Eꢀcadherin indicating the specificity of
our siRNA (Data not shown). These results clearly suggested that the attenuation of MDM2 by
C4 was responsible for abrogation of EMT. Next, we sought to identify whether overexpression
of MDM2 could have any relevance with the process of metastasis or not and whether treatment
with C4 could have the similar effects on metastasis in the MDM2 overexpression condition or
as in the condition mimicking MDM2 basal expression. To achieve this, MDAꢀMBꢀ231 and
PCꢀ3 cells were transfected with pCMVMDM2/pCMV expression plasmid alone or in
combination with 3µM concentration of C4 (Figure 5B). Ectopic MDM2, not the pCMV vector
significantly reduced the Eꢀcadherin expression. This reduction was restored upon treatment with
3µM of C4 indicating the novelty of this molecule to modulate Eꢀcadherin. MDM2
overexpression also sparked the concomitant upregulation of Snail, Twistꢀ1 and Vimentin. Our
western blot results for knockdown and overexpression of MDM2 were further corroborated with
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immunocytochemistry experiments in PCꢀ3 cells. C4 was clearly able to undermine the ectopic
MDM2 (Figure 5C). Taken together, our results indicate that C4 imitated the same manner as
knockdown of MDM2 in diverse cancer cells and the overexpression of MDM2 led to a severe
metastatic phenotype, which could be reversed by C4 exposure as was evident from the bright
field images (Supporting Figure S2A). Further, we performed the gelatin matrix degradation
assay to investigate the effects of MDM2 overexpression / knockdown on the invasion potential
of PCꢀ3 cells (Figure 5D).The results showed that in comparison to the vehicle, the percent
threshold area of gelatin degradation was enhanced by 90% in the MDM2 overexpressed
condition. In contrast, the percent degradation area was reduced by 73.8 and 67.5 % in C4 and
siRNA MDM2 treated conditions, respectively (Supporting Figure S2B), while MDM2
overexpression facilitated a more aggressive and invasive phenotype, the reverse was observed
in case of MDM2 knockdown and C4 treated conditions. Besides, C4 was also able to overcome
the effects of MDM2 overexpression on matrix gelatin degradation to a significant extent.
Almost, a 65 % decrease was observed in the condition of MDM2 overexpression along with C4
treatment compared to alone MDM2 overexpression condition. Therefore, taken together,
abrogation of metastasis by C4 is primarily due to its inhibitory effect on MDM2.
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In vivo potential of C4
Further, to determine the in vivo efficacy of C4, the 4T1 spontaneous/orthotopic mouse
mammary carcinoma model was employed. A doseꢀresponse profile for C4 was performed prior
to the administration of C4 to tumor bearing animals. A dose of 20 mg/kg b.w. was found to be
tolerable and nonꢀtoxic and was selected for further experimentation (Supplementary Table S1).
The average body weights of control and C4 treated mice were monitored throughout the study.
In this model, following primary tumors grown within the mammary pad of the mice, the mice
were treated with 20 mg/kg b.w. of C4, negative control (normal saline) and 5ꢀFU (positive
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control) on each alternate day for two weeks. Following completion of the treatment, the animals
were sacrificed; the tumor and lungs were dissected and evaluated for tumor growth inhibition
(Figure 6A) as well as suppression of metastatic lung nodules, respectively.
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Figure 6. C4 inhibits primary tumor growth and prevents lung metastasis. (A) Mouse mammary
carcinoma (4T1) cells were allowed to form the tumor subcutaneously beneath the second
mammary pad of Balb/c mice and subsequently treated with 20mg/kg b.wt. of C4 for two weeks.
5ꢀFU (20mg/kg b.wt.) was used as a positive control. Animals were sacrificed thereafter and
tumor growth was measured. (B and C) represent the tumor weight (mg) and tumor volume
(mm3), respectively. (D) Lungs of the animals were then dissected carefully and observed under
an inverted microscope for the formation of metastatic lung nodules. The number of lung
nodules is given in the form of a bar graph. Data is representative of three independent
experiments performed prior to the statistical analysis. * P<0.05**P<0.01, ***P<0.001.
We observed a considerable reduction (52%) in the primary tumor weight of C4 treated group as
compared to the control group (Figure 6B). 5ꢀFU was used as a positive control and showed a
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reduction of 64% in the tumor weights as compared to the control group. Correspondingly, the
tumor volume was reduced by 67% in 5ꢀFU treated group and 65% in C4 treated group (Figure
6C). The percent reduction in the number of metastatic nodules was 75% and 65% in 5ꢀFU and
C4 treated groups, respectively (Figure 6D).Taken together, these results confirm the
antimetastatic potential of C4 in vivo in breast cancer model at a safe and tolerable dose of 20
mg/kg b.w.
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Discussion and conclusion
In order to explore the MDM2 inhibitors, we investigated the conjugates of c(LysꢀPro) and
c(OrnꢀPro) with piperic acid and 4ꢀethylpiperic acid. The development of conjugates is based on
the cyclodipeptide 52RR4 in which we conjugated the side chain of Lys/Orn of cyclic dipeptides
with piperic acid/4ꢀethylpiperic acid. The Lys and Orn residues were used in the peptides to
conjugate with piperic acid/ 4ꢀethylpiperic acids, while conformationally constrained residue
proline facilitates the cyclization of peptides. Cyclodipeptide 52RRhas been reported to show the
inhibition of p53/MDM2 interactions.4 Recently, we have reported that the conjugation of
piperic acid/ 4ꢀethylpiperic acid with βꢀamino acid, β3,3ꢀPip inhibits the invasion and migration
of PANCꢀ1 cells.29,30 The main barrier to the treatment of metastases is the biological
heterogeneity of cancer cells in the primary neoplasm and in metastases.31,32 This heterogeneity
results in an evasion mechanism and confers drug resistance against conventional therapeutic
regimens to cancer cells.33 Gene amplification and protein overexpression of MDM2, which is
often found in certain types of cancers, indicate that MDM2 plays an important role in
tumorigenesis.18,34 It confers to tumor cells the ability to bypass apoptosis, facilitating tumor
growth and progression.35 Therefore, MDM2 mediated resistance to chemotherapy is a
provocative challenge to new cancer drug development.36, 37 Here, we have underscored the
relevance of peptide conjugate C4 on suppression of metastatic spreading of cancer cells to
distant organ/s facilitating secondary tumor formation. This conjugate not only abrogated the
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MDM2ꢀdependent cancer cells invasion but also terminated aggressive mouse mammary
carcinoma growth and metastasis in vivo. Interestingly, over the last decade or so, apart from the
p53 regulatory roles of MDM2, other nonꢀcanonical functions of this protein are emerging.
MDM2 expression has been positively correlated with a higher degree of metastasis and poor
prognosis in clinical patients.9,38 In pancreatic cancer cell model, a microarray based
investigation revealed that MDM2 was upꢀregulated along with 39 other metastasisꢀrelated
genes, along with 13 ECMꢀrelated genes.39 Importantly, MDM2 also triggers the ubiquitination
of Eꢀcadherin40, leading to the downꢀregulation of Eꢀcadherin protein levels and Eꢀcadherin
mediated cellꢀcell contacts. Wang et al. found a statistically significant correlation between
MDM2 overexpression and Eꢀcadherin downregulation in tumor specimens recovered from
human breast cancer patients with lymph node metastases.26 Herein, we demonstrate that C4
leads to the upregulation of Eꢀcadherin with concomitant downꢀmodulation of key mesenchymal
markers (Snail, Twistꢀ1, Vimentin) to attenuate metastasis. Previously, it has been reported that
p53 controls cancer cell invasion by inducing the MDM2ꢀmediated degradation of Snail.41
Similarly, reciprocal effect of C4 on Snail (which is a negative repressor of Eꢀcadherin and an
invasion promoting factor) was observed in both the cell lines tested in this report. Moreover,
Twistꢀ1 is another key protein involved in the metastatic nature of cancer cells and is upstream of
Snail. It binds to the promoter of Snail, thereby, acting as its coꢀtranscriptional activator and
drives the expression of Snail.27 Of note, C4 also resulted in the downregulation of Twistꢀ1 in a
dose dependent manner. Furthermore, expression levels of MMP2 and MMP9, a class of
enzymes responsible for remodeling the extracellular matrix, were also down modulated by C4
in a dose dependent manner. This is in accordance with the already established link between
MMP9 and MDM2.42 A mouse xenograft tumor model that over expressed betaꢀarrestin, a
known regulator of MDM2, showed increased MMP9 activity with more aggressive tumors.43
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Further, our MDM2 overexpression and knockdown studies using breast and prostate cell lines
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showed a direct link between MDM2 downregulation and the suppression of metastatic
phenotype. In addition, our in vivo results with C4 exhibited a significant reduction in the
primary tumor weight and metastasis of 4T1 cells to the lungs employing 4T1 metastatic model.
Overall, this study advocates C4 a potential lead molecule with significant cytotoxic and antiꢀ
metastatic activity operating through a MDM2 dependent mechanism.
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Experimental Section
Chemistry
All the reagents were obtained from commercial sources and used without purification. The tertꢀ
butoxycarbonyl (Boc) group was used for Nꢀterminal protection, while Cꢀterminal was protected
as methyl ester. Deprotection of Boc and methyl ester was performed using 30% trifluoroacetic
acid (TFA) in dichloromethane (DCM) and saponification, respectively. The coupling reactions
were performed by using 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide hydrochloride
(EDC.HCl) in the presence of Nꢀmethylmorpholine (NMM). Thin layer chromatography was
used to monitor the progress of reaction. The purification was performed by column
chromatography using silica gel (60ꢀ120 mesh) as stationary phase and MeOH /CHCl3 as mobile
phase. The final compounds were purified using high performance liquid chromatography
(HPLC) on reverse phase Cꢀ18 column (10mm × 250mm) using methanol/water gradients. The
characterization of all the peptides and conjugates achieved by HRMS having QTOF analyser
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(Agilent Technologies 6540), H and C NMR spectra, which were recorded on a Bruker 500
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(500.13 MHz for H; 125.76 MHz for C) either in CDCl3 or DMSOꢀd6. The chemical shift (δ)
values are reported in parts per million (ppm). The coupling constant (J) values are given in
hertz (Hz).
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoic acid, piperic acid(PA)
Piperic acid was prepared according to the procedure reported in the literature.19 Piperine was
dissolved in ethylene glycol and potassium hydroxide and refluxed at 1800C. The completion of
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reaction was monitored by thin layer chromatography. After completion of reaction, the reaction
mixture was diluted with ice cold water followed by acidification with 2NꢀHCl. The resulting
precipitate was filtered, washed with water and dried to yield crude product, which was
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crystallized in ethanol to afford piperic acid. Yield: 80%. H NMR (500 MHz, DMSO): δ 7.27
(dd, J =15.3, 8.9Hz, 2H), 7.00 (d, J=7.8 Hz, 1H), 6.94 (m, 3H), 6.05 (s, 2H), 5.94 (d, J=15.2 Hz,
1H).13C NMR (126 MHz, DMSO): δ 167.95, 147.97, 147.93, 143.87, 139.25, 130.56, 124.98,
122.97, 122.15, 108.46, 105.62, 101.31. HRMS (ESI): MCal=218.21; MObs=219.32 [M+H]+.
(2E,4E)-4-(benzo[d][1,3]dioxol-5-ylmethylene)hex-2-enoic acid, 4-ethylpiperic acid (EPA)
4ꢀEthylpiperic acid was synthesized by literature procedure.30 Piperonal was used as the starting
material to carry out Grignard reaction using ethyl iodide and magnesium at 00C under N2 to
yield 1ꢀ(benzo[d][1,3]dioxolꢀ5ꢀyl)propanꢀ1ꢀol, which on treatment with Vilsmeier reagent
(DMF/POCl3) resulted in the formation of an unsaturated aldehyde, (E)ꢀ2ꢀ(benzo[d][1,3]dioxolꢀ
5ꢀylmethylene)butanal. The Wittig reaction of (E)ꢀ2ꢀ(benzo[d][1,3]dioxolꢀ5ꢀylmethylene)butanal
with ylide salt prepared from triphenylphosphine and ethyl bromoacetate gave (2E,4E)ꢀethylꢀ4ꢀ
((benzo[d][1,3]dioxolꢀ6ꢀyl)methylene)hexꢀ2ꢀenoate. The saponification of (2E,4E)ꢀethylꢀ4ꢀ
((benzo[d][1,3]dioxolꢀ6ꢀyl)methylene)hexꢀ2ꢀenoate afforded (2E,4E)ꢀ4ꢀ(benzo[d][1,3]dioxolꢀ5ꢀ
ylmethylene)hexꢀ2ꢀenoic acid, which was further crystallized in ethyl acetate to yield colorless
1
crystalline solid of 4ꢀethylpiperic acid. Yield:1.3g (72 %). H NMR (500 MHz, CDCl3): δ 7.09
(d, J =15.7 Hz, 1H), 6.63 (s, 1H), 6.59 (d, J = 7.9Hz, 2H), 6.42 (s, 1H), 5.77 (s, 2H), 5.71 (d, J =
13
15.7 Hz, 1H), 2.27 (q, J = 7.5 Hz, 2H), 0.96(t, J=7.5 Hz, 3H). C NMR (126 MHz, CDCl3): δ
173.71, 153.55, 152.65, 152.15, 143.58, 142.50, 135.21, 128.53, 122.01, 113.55, 113.21, 106.12,
24.98, 18.11. HRMS (ESI): MCal = 246.26; MObs= 247.27 [M+H]+.
General Procedure for the Synthesis of c(Lys-Pro), P1 and c(Orn-Pro), P2
4.91g (10mmol) of BocꢀLys(Cbz)ꢀOH/BocꢀOrn(Cbz)ꢀOH was dissolved in 5ml of dry
dichloromethane at ice cold temperature and added LꢀProꢀOMe. HCl (10mmol, 1.29g) and
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EDC.HCl (11.0mmol, 2.1g) in presence of NMM (2.5ml). After completion of reaction of
reaction, the solvents were evaporated from the reaction mixture, added 10ml of water and
extracted with ethyl acetate (3x10ml). The organic layer was washed with 2NꢀHCl (1x10ml),
1MꢀNa2CO3 (1x10ml) and brine solution (1x10ml). The organic layer was dried over anhydrous
Na2SO4 and evaporated on rotavapor to yield dipeptides, BocꢀLys(Cbz)ꢀProꢀOMe/Bocꢀ
Orn(Cbz)ꢀProꢀOMe, which was purified by column chromatography using silica gel (100ꢀ
200mesh) as stationary phase and chloroform/methanol as mobile phase.
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2.4g (5mmol) of dipeptides, BocꢀLys(Cbz)ꢀProꢀOMe/ BocꢀOrn(Cbz)ꢀProꢀOMe were
deprotected with 30% TFA in CH2Cl2 for 3h. The solvent was removed in vacuum and the
deprotected dipeptides were dissolved in minimum amount of dry DMF and 2.0 eq. of piperidine
to facilitate cyclization. The reaction mixture was stirred at room temperature for 2h. The solvent
was removed completely and the resulting compound was purified by column chromatography
using silica gel (60ꢀ120 mesh) and with chloroform/methanol to give the dipeptides, c[Lys(Cbz)ꢀ
Pro], and c[Orn(Cbz)ꢀPro]. Finally, the carboxybenzyl (Cbz) group was deprotected by using
10% Pd/C in dry methanol under H2 for 12h to yield the cyclic dipeptides c(LysꢀPro), P1 and
c(OrnꢀPro), P2.
1
c(Lys-Pro), P1: White crystalline solid, yield: 0.700g (75%). H NMR (400 MHz, DMSO): δ
8.31 (d, J = 4.0 Hz, 1H), 4.28 – 4.11 (m, 2H), 4.04 (t, J = 9.2 Hz, 1H), 3.68 – 3.60 (m, 1H), 2.76
(m, 2H), 2.22 – 2.07 (m, 2H), 1.88 – 1.83 (m, 2H), 1.79 (dd, J = 10.7, 5.6 Hz, 2H), 1.68 – 1.63
(m, 2H), 1.58 (d, J = 7.7 Hz, 2H), 1.41 – 1.36 (m, 2H). 13C NMR (101 MHz, DMSO): δ 170.48,
168.96, 58.96, 57.02, 54.65, 45.48, 33.45, 29.05, 26.92, 22.68, 22.13, HRMS (ESI): MObs
=
225.147; MCal= 226.154[M+H]+.
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c(Orn-Pro), P2: White crystalline solid; yield: 0.675g (60%). H NMR (500 MHz, DMSO) δ
8.38 (d, J = 3.2 Hz, 1H), 4.22 – 4.15 (m, 2H), 4.11 (s, 1H), 3.63 (q, J = 5.2Hz, 1H), 2.77 (m,2H),
2.18 – 2.11 (m, 2H), 1.88 – 1.84 (m, 2H), 1.80 (d, J = 8.3 Hz, 2H), 1.74 – 1.71 (m, 2H), 1.68 –
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1.64 (m, 2H).13C NMR (101 MHz, DMSO) δ 168.96, 165.96, 57.95, 56.88, 45.52, 45.25, 30.91,
28.97, 28.15, 26.19, 23.79. HRMS (ESI): MObs= 211.132; MCal=212.140[M+H]+.
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General Procedure for the Synthesis of C1- C4
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1mmol of piperic acid/4ꢀethylpiperic acid was dissolved in dry dichloromethane (1ml) and
cooled in an ice bath while stirring. NMM (2.0ml) and EDCI.HCl (2mmol, 0.382) were added to
the reaction mixture. After that 1mmol of c(Lys/OrnꢀLꢀPro) was added into the reaction mixture
and stirred for 12h. Progress of the reaction was monitored using TLC at regular intervals. After
completion of the reaction, the solvent was evaporated and the residue dissolved in ethyl acetate.
The organic layer was washed successively with 2NꢀHCl (3×10 ml), 2MꢀNa2CO3 (3×10 ml), and
brine. The combined organic layer was dried over anhydrous sodium sulphate and evaporated in
vacuum to yield PA/EPAꢀCyclo(Lys/OrnꢀLꢀPro),which was purified by column chromatography
over silica gel (60ꢀ120 mesh). The purity of all the conjugates was checked by high performance
liquid chromatography.
PA-c(Lys-Pro), C1: Yield: 0.250g (50%).1H NMR (400 MHz,CDCl3): δ 7.37ꢀ7.30 (m, 1H), 6.98
(s, 1H), 6.89 (d, J = 7.6 Hz, 1H), 6.79 (d,J=8.4Hz,1H) 6.69 (d, J = 11.2 Hz, 1H), 6.41 (s, 1H),
5.98 (s, 2H), 5.93 (d, J = 8 Hz, 1H), 5.89 (d, J = 8 Hz, 1H), 5.73 (s, 1H), 4.09 (m, 1H), 3.90 (m,
1H), 3.63 – 3.50 (m, 3H), 3.41 – 3.32 (m, 2H), 2.47– 2.31(m, 2H), 2.10(m,3H), 1.97–
1.85(m,3H). 13CNMR (126MHz, CDCl3): δ 169.34, 166.40, 165.85, 148.24, 141.50, 141.22,
139.13, 130.84, 124.63, 122.93, 122.67, 108.51, 105.73, 101.31, 59.18, 58.14, 45.63, 39.16,
29.08, 28.18, 22.61, 22.13. HRMS (ESI): MObs= 425.195; MCal =426.202[M+H]+.
PA-c(Orn-Pro), C2: Yield: 0.240g(58%). 1H NMR (400 MHz, CDCl3): δ 7.38 (s, 1H), 7.31 (d,
J =14.8 Hz 1H), 6.95 (s, 1H), 6.87 (dd, J = 8.1, 1.4 Hz, 1H), 6.77 (s, 1H), 6.74 (d, J = 5.6 Hz,
1H), 6.63 (d, J = 15 Hz 1H), 6.44 (t, J = 10.4 Hz 1H), 5.97(s, 2H), 5.94(s, 1H), 4.13 (d, J = 7.2
Hz, 1H), 4.10 (s, 1H), 3.6ꢀ3.53 (m, 2H), 3.44ꢀ3.35 (m, 2H),2.42ꢀ2.32(m,1H),2.08ꢀ
1.99(m,3H),1.73(m,2H),1.30ꢀ1.16(m,2H).13CNMR(126MHz,CDCl3):
δ172.10(s),
168.13,
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167.25, 149.62, 142.44, 140.34, 132.25, 126.01, 124.59, 124.08, 109.84, 107.11, 102.74, 60.62,
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56.48, 46.85, 40.45, 29.70, 27.76, 26.18, 24.11. HRMS (ESI): MObs=411.179; MCal
412.186[M+H]+.
=
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EPA-c(Lys-Pro), C3: Yield:0.250g (57%). 1H NMR (400 MHz, CDCl3): δ 7.37 – 7.29 (m, 1H),
6.85 (s, 1H), 6.82 (s, 1H), 6.67 (d, J = 11.2 Hz, 1H), 6.55 (s, 1H), 5.98 (s, 2H), 5.96 (d, J = 1.6
Hz 1H), 5.95 – 5.90 (m, 1H), 5.82 (t, J = 7.2 Hz, 1H), 4.13 – 4.06 (m, 1H), 3.91 (m, 1H), 3.61 –
3.51 (m, 2H), 3.38 (m, 2H), 2.49 (q, J = 7.6 Hz, 2H), 2.44 – 2.32 (m, 2H), 2.08 – 1.98 (m, 2H),
1.94 – 1.84 (m, 2H), 1.62 (m, 2H), 1.53 – 1.45 (m, 2H), 1.18 (t, J = 10.2 Hz, 3H). 13C NMR
(101 MHz, CDCl3)δ173.16, 168.36 ,165.83,147.76, 147.08, 145.49,138.86, 130.97, 123.61,
118.86, 108.86 ,108.38, 101.18 , 57.81 , 55.15, 45.36, 39.22 , 33.83, 29.21, 28.19, 22.70, 22.11,
21.50, 20.43, 13.45. HRMS (ESI): MObs=453.226; MCal =454.232[M+H]+.
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EPA-c(Orn-Pro), C4:Yield:0.300g (68%): H NMR (400 MHz, CDCl3): δ 7.30ꢀ7.22 (m, 1H),
6.99 (d, J = 14.8 Hz, 1H), 6.83 (s, 1H), 6.80 (d, J = 0.9 Hz, 2H), 6.63 (s, 1H), 6.22ꢀ 6.11 (m, 1H),
5.96 (s, 2H), 5.92 (dd, J = 15.4, 7.3 Hz, 1H), 4.11ꢀ4.07 (m, 1H), 3.98 (dd, J = 10.6, 7.2 Hz, 1H),
3.69 – 3.49 (m, 2H), 3.48 – 3.28 (m, 2H), 2.46 (q, J = 7.5 Hz, 2H), 2.40 – 2.33 (m, 1H), 2.08 –
1.98 (m, 2H), 1.92 (m, 2H), 1.77 – 1.70 (m, 2H), 1.15 (t, J = 12 Hz, 3H). 13C NMR (101 MHz,
CDCl3) δ 170.30, 169.13 , 166.80, 147.79, 147.14 , 145.39, 138.89, 130.93, 123.59, 118.78,
108.84, 108.37, 101.17, 59.17, 58.16, 45.63, 38.89, 31.84, 29.09, 28.26, 25.67, 22.65, 20.42,
13.41. HRMS (ESI): MObs=439.210; MCal = 440.217[M+H]+.
Biology
Cell Culture and Reagents
Cell lines used were purchased from American Type Cell Culture (ATCC). Cells were cultured
in their respective specified media (RPMI 1640, DMEM, Lꢀ15 supplemented MEM) containing
10% fetal bovine serum (Invitrogen, Carlsbad, CA). Paraformaldehyde, Phenylmethylsulfonyl
fluoride (PMSF), Dithiothreitol (DTT), NPꢀ40, Protease Inhibitor Cocktail, Dimethylsulfoxide
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(DMSO) and Bradford reagent were bought from Sigma Chemicals Co. (St. Louis, MO),
UltraCruz DAPI (4,6ꢀdiamidinoꢀ2ꢀphenylindole) mounting medium was obtained from Santa
Cruz Biotechnology Inc. (Santa Cruz, CA). C4 and other conjugates were synthesized in house at
IIIM, Jammu and dissolved in DMSO. The final concentration of DMSO in the experiment never
exceeded 0.2%.
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Cell Viability Assay
The cell viability was determined by standard MTT assay method. Briefly, cancer cell linesꢀ
MDAꢀMBꢀ231, MCF7, PC3, HCTꢀ116, MiaPacaꢀ2, and normal breast epithelialꢀfR2 cells were
seeded in 96 well tissue culture plates (Nunc) at a density of 3.5 × 103 cells per well and treated
with varying concentrations of the test conjugates 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
44h of incubation, MTT dye solution was added into the medium and cells were incubated for
another 4 h at 37 °C in 5% CO2and humidified environment. The amount of coloured formazan
crystals formed was measured by taking optical density (OD) using a microplate reader
(TECAN, Infinite M200 Pro) at 570 nm and the percentage cell viability was calculated. The
IC50 values were calculated using GraphPad Prism software (Version 5.0).
Immunoblotting
Cells (0.5×106) were seeded in six well plates for overnight in a humidified CO2 incubator and
then exposed to varying concentrations of C4 for 24h along with vehicle. Cells were then
harvested, washed with chilled PBS and lysed with lysis buffer (HEPES 1 mM, KCl 60 mM, NPꢀ
40 0.3%, EDTA 1 mM, DTT 1 mM, sodium orthovanadate 1mM, PMSF 0.1mM and cocktail
protease inhibitor). The cell lysates were centrifuged at 12,000 rpm for 20 min at 4°C. Protein
concentration was measured by the standard Bradford method. An equal amount (30ꢁg) of
protein from each sample was subjected to SDSꢀPAGE, and proteins were transferred to PVDF
membranes (PALL), blocked with 5% (w/v) nonꢀfat milk in PBS or 5% (w/v) BSA in PBS
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containing 0.1% Tweenꢀ20, probed with specified antibodies for 3 h at room temperature or
overnight at 4 °C, and subsequently washed and probed with speciesꢀspecific secondary antibody
coupled to horseradish peroxidase (HRPO). Immunoꢀreactive proteins were detected by
Enhanced Chemiluminescence Plus (Amersham).
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Scratch Motility (Wound Healing) Assay
According to the preꢀstandardized procedure44, MDAꢀMBꢀ231 and PCꢀ3 cells were plated in
6ꢀwell plates at a concentration of (1×106 cells/well) and allowed to form a confluent monolayer
for 24h. On the next day, the monolayer was scratched with a sterile pipette tip (20ꢀ200ꢁl). To
remove the detached cells, washing was done with serum free medium. Subsequently, cells were
treated with varying concentrations of C4, and vehicle, in starvation medium for another 24h.
Wounded areas were progressively photographed under an inverted microscope with Nikon
D3100 camera at 20x magnification. The average number of migratory cells per field was
counted under a microscope.
Cell Scattering Assay
The cell scattering assay was performed as described previously.45 Briefly, cells were seeded at
a density of 1000 cells/well in 6ꢀwell plates. Small, cohesive, and discrete colonies were formed
after 3ꢀ4days of incubation. Cells were then stimulated with VEGF (20ng/ml) in the presence of
various concentrations of the C4 diluted in growth medium. After overnight incubation, colonies
were fixed and stained with 0.2% crystal violet. Photographs were taken for individual colonies.
Fluorescent Gelatin Degradation Assay
Crossꢀlinkedꢀfluorophore (FITC)ꢀconjugated gelatin matrixꢀcoated coverslips were prepared
according to standardized protocol.45Coverslipscoated with gelatin were quenched with RPMI
containing 10% fetal bovine serum at 37 ºC for 60 minutes prior to seeding of cells. To assess the
formation of podosomes or invadopodia and degradation of FITCꢀgelatin matrix, MDAꢀMBꢀ231
and PCꢀ3 cells were cultured on FITC gelatinꢀcoated coverslips in the density of 0.1×106 cells/
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coverslip. After 16 h, cells were treated with different concentrations of C4 for 24 h followed by
DAPI mounting media and then observed under FLoid Cell Imaging Station for determining
gelatin degradation. Images were further processed for determining the threshold area of
degradation by ImageꢀJ software.
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Matrigel Invasion Assay
The effect of C4 treatment on cell invasion was determined using BD Biocoat Tumor Invasion
Assay System (BD Bioscience, Bedford, MA) according to the instruction of the manufacturer.
Briefly, MDAꢀMBꢀ231 and PCꢀ3 cells (1.2×106) cells were cultured in the presence of different
indicated concentrations for 24 h in serum free media into the upper chambers/inserts, and the
bottom wells were filled with chemoꢀattractant (complete media with 10% FBS). Cells were
allowed to migrate at 37°C. After 24 h, the Matrigelꢀcoated polycarbonate filters were removed,
the nonꢀmigrating cells were separated 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.
Gelatin Zymography
The gelatinase activity of MMPꢀ2 and MMPꢀ9 was analyzed by method standardized
previously.45 Accordingly, MDAꢀMBꢀ231 and PCꢀ3 cells in subꢀconfluent culture (70%
confluency) were transfected and/or treated with indicated conditions. The conditional media
obtained from both treated and untreated samples were employed for protein estimation and an
equal amount of total proteins (20 µg) were mixed with sample buffer (2.0% SDS, 25% glycerol,
0.1% Bromophenol blue and 60mM TrisꢀHCl, pH 6.8). Gelatin zymography of the samples was
carried out using 7.5% SDSꢀpolyacrylamide gels containing 0.1% gelatin at 100V for 3 h
followed by routine commasie staining. Gelatinase activity was detected by observing unstained
bands on a blue background of comassie stained gel.
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Plasmids and transfections
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Plasmids expressing pCMV MDMꢀ2 or pCMV backbone were a kind gift from Dr. Mir Sajad
(University of Kentucky, Lexington USA). Transfections were carried out with Lipofectamine
3000 (Thermo Fisher Scientific) or Neon Transfection system (Invitrogen Life Technologies) as
per the manufacturer’s protocol and as described previously.45
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siRNA transfection
MDM2 siRNA was purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Briefly, the
desired number of cells was seeded in appropriate dishes and transfected with human specific
Parꢀ4. Lipofectamine 3000 (Thermo Fisher Scientific) was used for transfections as per the
manufacturer's guidelines.
Immunocytochemistry
For MDM2 protein immunostaining, the cells were seeded in the LabꢀTek chamber slides
(Thermo Scientific, Boston, MA, USA) at a seeding density of 5 × 104 cells/chamber. Following
transfections or treatments with C4, the cells were incubated for set time points and
immunocytochemistry was performed. The prepared slides were then analyzed with EVOS
FLoid Cell Imaging Station (Thermo Fisher Scientific) or with Zeiss LSMꢀ510 metaconfocal
microscope under ×40 magnification (scale used was 100ꢁM unless otherwise specified).
Mouse 4T1 breast tumor model
Prior to the treatment of C4 to the tumor bearing animals, a doseꢀresponse study was carried out
to determine the safety/tolerable dose of C4. It was administered into the normal Balb/c mice
(without tumor) at increasing doses, i.e. 5, 10, 15, 20, 30, 50 and 100mg/kg, b.w. to seven
separate group of animals. The doses were given intraperitoneally in each alternate day for seven
days and animals were observed for any deleterious effects. Further, to evaluate the in vivo
antitumor and antimetastatic effect of C4, healthy female Balb/c mice (b.w. 20ꢀ30 g) were taken.
All the in vivo experimental protocols were approved by the Institutional Animal Ethics
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Committee, CPCSEA, of Indian Institute of Integrative Medicine, Jammu. The animals were
maintained at 22°C with a 12h light dark cycle and free access to feed and water inside the
institutional animal house. Proper care was taken to maintain them in a healthy condition and to
avoid any risk of possible pathogenic contaminations. For subcutaneous implantation, the mice
were randomized into three groups of 5 mice per group and 1.5million 4T1 cells in serum free
RPMI media were injected subcutaneously into the mammary pad of each mouse. Either 1 week
after the tumor cell implantation or when the tumor volume reached 30ꢀ150 mm3, mice were
injected intraperitoneally with either vehicle (normal saline) or C4 (20mg/kg/b.w.), every
alternate day for two weeks. 5ꢀFU (20 mg/kg/ b.w.), was used as a positive control. Tumor size
was recorded on each alternate day postꢀcell injection and body weights were recorded once per
week. Mice were sacrificed 15 days after the treatment initiation and tumors were incised. Tumor
dimensions were measured using a standard Vernier caliper. Both tumor volume as well as tumor
weight were recorded. Metastatic lung nodules were counted using a dissecting microscope.
Statistical Analyses
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Data were expressed as mean ± SEM of at least three independent experiments performed. No
statistical method was used to predetermine sample size. The experiments were executed and
analyzed in a nonrandomized and nonꢀblinded manner. Comparisons between two groups were
conducted using unpaired Student’s tꢀtest. * P<0.05** P<0.01and***P<0.001values were
assigned significance.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website via the
Internet at http://pubs.acs.org
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