Pyrimidine-2,4-dione targets STAT3 signaling pathway to induce cytotoxicity in hepatocellular carcinoma cells

Article abstract of DOI:10.1016/j.bmcl.2021.128332

Signal transducer and activator of transcription 3 (STAT3) is a tumorigenic transcription factor that is persistently activated in various human cancers including hepatocellular carcinoma (HCC). Therefore, STAT3 is considered as a prominent target to counteract the uncontrolled proliferation of cancer cells. In the present report, pyrimidine-2,4-diones (N-methyluracil derivatives) (MNK1-MNK14) were synthesized in an ionic liquid (BMIm PF₆) medium employing a ligand-free Suzuki-Miyaura cross-coupling process. Among the 14 derivatives, compound MNK8 showed good cytotoxicity towards both the tested cell lines and did not display a toxic effect against normal hepatocytes (LO2). MNK8 significantly increased the Sub-G1 cell count in both cell lines and the cytotoxic effect of MNK8 was found to be mediated through the suppression of constitutive phosphorylation of STAT3^Y705. It also decreased the DNA interaction ability of nuclear STAT3 in HCC cells. MNK8 downregulated the levels of apoptosis-related proteins (such as Bcl-2, cyclin D1, survivin) and increased cleaved caspase-3 inferring the apoptogenic effect of MNK8. It also reduced the CXCL12-triggered cell migration and invasion in in vitro assay systems. Overall, MNK8 has been demonstrated as a new inhibitor of STAT3 signaling cascade in HCC cells.

Full text of DOI:10.1016/j.bmcl.2021.128332

Bioorg. Med. Chem. Lett. 50 (2021) 128332
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrimidine-2,4-dione targets STAT3 signaling pathway to induce
cytotoxicity in hepatocellular carcinoma cells
,
,
Ayyiliath M Sajitha 1, Kereyagalahally H. Narasimhamurthyb 1, Muthu K. Shanmugamc,
Shobith Rangappad, S. Chandra Nayake, Arunachalam Chinnathambif,
Tahani Awad Alahmadig, Sulaiman Ali Alharbif, K.R. Haridasa, E.K. Reddyh, B. Savithai,
Chakrabhavi Dhananjaya Mohanj *, Kanchugarakoppal S. Rangappae
,
,*
a School of Chemical Sciences, Kannur University, Payyanur Campus, Edat P.O., Kannur 670327, India
b Department of Studies in Organic Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India
c Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117600, Singapore
d Adichunchanagiri Institute for Molecular Medicine, Adichunchanagiri, BG Nagara 571448, Nagamangala Taluk, India
e Institution of Excellence, Vijnana Bhavan, University of Mysore, Manasagangotri, Mysore 570006, India
f Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
g Department of Pediatrics, College of Medicine, King Saud University, [Medical City], King Khalid University Hospital, PO Box-2925, Riyadh 11461, Saudi Arabia
h Department of Chemistry, Vignan’s Foundation for Science and Technology and Research, Vadlamudi, Guntur 522213, India
i Postgraduate and Research Department of Chemistry, Jamal Mohamed College, Bharathidasan University, Tiruchirappalli 620020, India
j Department of Studies in Molecular Biology, University of Mysore, Manasagangotri, Mysore 570006, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Signal transducer and activator of transcription 3 (STAT3) is a tumorigenic transcription factor that is persis-
tently activated in various human cancers including hepatocellular carcinoma (HCC). Therefore, STAT3 is
considered as a prominent target to counteract the uncontrolled proliferation of cancer cells. In the present
report, pyrimidine-2,4-diones (N-methyluracil derivatives) (MNK1-MNK14) were synthesized in an ionic liquid
(BMIm PF6) medium employing a ligand-free Suzuki-Miyaura cross-coupling process. Among the 14 derivatives,
compound MNK8 showed good cytotoxicity towards both the tested cell lines and did not display a toxic effect
against normal hepatocytes (LO2). MNK8 significantly increased the Sub-G1 cell count in both cell lines and the
cytotoxic effect of MNK8 was found to be mediated through the suppression of constitutive phosphorylation of
STAT3Y705. It also decreased the DNA interaction ability of nuclear STAT3 in HCC cells. MNK8 downregulated
the levels of apoptosis-related proteins (such as Bcl-2, cyclin D1, survivin) and increased cleaved caspase-3
inferring the apoptogenic effect of MNK8. It also reduced the CXCL12-triggered cell migration and invasion in
in vitro assay systems. Overall, MNK8 has been demonstrated as a new inhibitor of STAT3 signaling cascade in
HCC cells.
STAT3 inhibitor
N-methyluracil derivatives
Anticancer
Antimigratory agent
Introduction
oncogenic protein that is overactivated in many types of human neo-
plasms including cancers of the pancreas, head and neck, colon, liver,
Signal transducer and activator of transcription (STAT) proteins are a
group of transcription factors comprising of seven members, namely
STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT61–2. Among
the STAT variants, STAT1, STAT3, and STAT5 proteins are the most
studied transcription factors in relevance to cancer initiation and pro-
gression3–4. Among these three proteins, STAT3 is widely studied as an
breast, lung, gastric, leukemia, and lymphomas5–7. In the classical
pathway, IL-6 family cytokines interact with their corresponding
transmembrane receptors which lead to dimerization of gp1308–9. The
dimeric gp130 triggers the activation of the Janus kinases (JAKs) and in
turn, JAKs phosphorylate STAT3 proteins at tyrosine-705 (STAT3Y705
)
at the SH2 domain10–12. The activated monomeric STAT3 undergoes
* Corresponding authors.
E-mail addresses: cd.mohan@yahoo.com (C.D. Mohan), rangappaks@gmail.com (K.S. Rangappa).
1
These authors contributed equally.
https://doi.org/10.1016/j.bmcl.2021.128332
Received 21 May 2021; Received in revised form 13 August 2021; Accepted 15 August 2021
Available online 19 August 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

A.M. Sajith et al.
Bioorganic & Medicinal Chemistry Letters 50 (2021) 128332
Fig. 1. (A) Scheme for the synthesis of N-methyluracil derivatives using Suzuki coupling reaction. (B) Structure of N-methyluracil derivatives produced via Suzuki
coupling reaction using ionic liquid.
dimerization with subsequent transportation to the nucleus13–14. In the
nucleus, STAT3 dimer interacts with specific DNA sequences in the
promoter region of STAT3-target genes to induce their tran-
scription15–17. Many genes that are under the transcriptional control of
STAT3 encode the proteins responsible for proliferation, angiogenesis,
prosurvival, antiapoptosis, and metastasis18–20. The aberrant activation
of STAT3 in human cancers results in deregulated transcriptional ac-
tivity thereby sustained proliferative signals in the tumor micro-
environment21–22. Therefore, it is an effective approach to counteract
the activation of STAT3 in cancer cells to inhibit their growth.
demonstrated the use of water as a solvent for the C C bond formation
of chloro uracils under microwave irradiation27. In the present study, we
have synthesized N-methyluracil derivatives in ionic liquid (BMIm PF6)
medium using the Suzuki coupling reaction. Among the 14 compounds,
MNK8 showed a good growth-inhibitory effect towards HCC cells
through abrogating the activation of the STAT3 signaling pathway.
The use of ionic liquids as an alternative medium for performing
carbon–carbon bond-forming reactions using transition metal catalysis
has become a focus of current green chemistry research28. This is
attributed to the non-flammable, non-volatile, highly solvating, non-
coordinating, and high thermal stability of the ionic liquids29. More
importantly, the use of ionic liquids is known to stabilize the true Pd(0)
nanoparticles and thereby increase the catalytic turnover of the cross-
coupling process30. Herein, we report the use of ligand-free Suzuki-
Miyaura protocol for the carbon–carbon forming process. The use of a
ligand-free procedure thus expedites the purification process that can be
encountered while employing phosphine ligands. The formation of polar
phosphine oxide by-products usually can impede the purification pro-
cess when dealing with polar heterocyclic analogs as cross-coupling
partners31. The successful synthesis of (hetero)arylated uracil analogs
via a ligand-free carbon–carbon bond-forming process further expands
–
Pyrimidine derivatives and their structural analogs have contributed
immensely to the field of cancer therapeutics since the approval of 5-
fluorouracil for the treatment of human cancers. 5-fluorouracil im-
parts antitumor potential by blocking the activity of thymidylate syn-
thase, which is crucial for the synthesis of thymine nucleotide23
.
Thereafter, many studies have extensively demonstrated the antitumor
potential of pyrimidine derivatives. Particularly, pyrimidines conju-
gated with other bioactive heterocycles (such as pyrazole, triazole,
thiazole, etc.) were evaluated for anticancer activity against various
cancer cells and many of them presented thymidylate synthase as the
cellular target of these derivatives24–26. In our previous study, we have
2

A.M. Sajith et al.
Bioorganic & Medicinal Chemistry Letters 50 (2021) 128332
lines. The other compounds of the MNK series were found to have an
IC50 value higher than 50 µM and compounds with IC50 value more than
50 µM are considered inactive in this study. Among MNK1 and MNK8
compounds, MNK8 effectively reduced the number of viable cells in a
concentration- and time-dependent fashion in both the tested cell lines
(Fig. 2A). WP1066 (a STAT3 inhibitor) was used as a positive control37
which also displayed a dose/time-dependent decrease of viable cells
(Fig. 2B). Thereafter, the effect of MNK8 on the normal hepatocytes
(LO2 cells) was examined. MNK8 showed minimal cytotoxicity towards
LO2 cells demonstrating its selectivity towards cancer cells. Therefore,
MNK8 was chosen as a lead compound for further experiments.
MNK8 selectively mitigates the constitutive activation of STAT3 in HCC
cells: To understand the underlying molecular mechanism in MNK8-
induced-cytotoxicity, MNK8 (25 µM) treated HepG2 and HCCLM3
cells were examined for the expression of phosphorylated STAT3 and
total STAT3 using Western blotting. MNK8 effectively reduced the
phosphorylation of STAT3Y705 in a duration-dependent fashion in both
the tested cell lines (Fig. 3A and B). There was no significant variation in
the levels of total STAT3 protein. Since MNK8 possesses the structural
features of a pan-kinase inhibitor, its effect on STAT5 activation was
examined in HepG2 and HCCLM3 cells. Interestingly, MNK8 did not
alter the constitutive phosphorylation of STAT5 in both cell lines
(Fig. 3C and D) indicating that MNK8 shows specificity towards STAT3
activation. Additionally, WP1066, a known STAT3 inhibitor was used as
a positive control37 to demonstrate the depletion of STAT3 activation
upon the treatment with a small molecule (Fig. 3E and F).
MNK8 downregulates the DNA interaction potential of STAT3 in HCC
cells: The activated STAT3 interacts with the DNA consensuses named
interferon-stimulated response element (ISRE) and γ-interferon activa-
tion sequence (GAS) which are present in the promoter of STAT3 target
genes38–39. To evaluate the DNA interaction potential of STAT3 in
MNK8-treated cells, HepG2, and HCCLM3 cells were preincubated with
MNK8 (25 µM) for a given duration. Subsequently, nuclear extract of
these cells was prepared and STAT3-DNA interaction was studied using
an ELISA assay kit. The treatment with MNK8 for various time points
suppressed constitutive STAT3 activity in a time-dependent fashion and
thereby decreased the STAT3-DNA interaction (Fig. 3G).
MNK8 increases Sub-G1 cells in a time-dependent fashion: During
apoptosis, initiator caspases such as caspase-8 and/or caspase-9 undergo
activation and in turn activates effector caspase (procaspase-3) by pro-
teolytic cleavage. The active caspase-3 cleaves a broad range of proteins
in the cell including caspase-activated DNase (CAD), lamins, focal
adhesion kinases, etc. CAD chops the genomic DNA into oligonucleotide
fragments which results in cells with lesser DNA content and these cells
appear as Sub-G1 cell population when analyzed using flow cytometry.
Next, HepG2 and HCCLM3 cells were incubated with MNK8 (50 µM) for
different durations (24, 48, and 72 h) and stained the cells with propi-
dium iodide to study the allocation of cells in different phases of the cell
cycle. The treatment of HepG2 and HCCLM3 cells with MNK8 (50 µM)
for the various durations (24, 48, and 72 h) increased Sub-G1 cells to
39.3, 46.5, and 67.3%, respectively (Fig. 4A).
Fig. 2. (A) MNK8 was screened for its cytotoxic efficacy towards HepG2 and
HCCLM3 cells at different concentrations (0, 10, 25, and 50 µM) and various
durations (24, 48, 72 h). (B) WP1066 was used as a positive control and it was
screened for its cytotoxic efficacy towards HepG2 and HCCLM3 cells at indi-
cated concentrations (0, 10, 25, and 50 µM).
the substrate scope that can be appreciated via a ligand-free method in
ionic liquids. The reaction optimization conditions have been given in
supplementary information. Among the tested conditions, the use of
BMIm PF6 as a green medium resulted in a 45% yield and the same re-
action condition was used for the synthesis of title compounds (Fig. 1A).
The structure of N-methyluracil compounds (MNK1-MNK14) is provided
in Fig. 1B.
N-methyluracil derivatives selectively impart cytotoxicity in HCC cells:
Initially, all the new N-methyluracil derivatives (MNK1-MNK14) were
evalu`ated for their growth hampering efficacy against HepG2 and
HCCLM3 cells using MTT assay as abnormal growth regulated by
different transcription factors is one of the key hallmarks of cancer
cells32–36. HepG2 and HCCLM3 cells were treated with new compounds
at various concentrations (0, 10, 25, and 50 µM) for the given duration
(24, 48, and 72 h). Among the tested compounds, MNK1 and MNK8
were found to be cytotoxic towards HepG2 (IC50: 47.6 and 20.9 µM
respectively) and HCCLM3 (IC50: 41.5 and 42.6 µM respectively) cell
MNK8 decreases the expression of antiapoptotic proteins: Functionally,
the majority of STAT3 target genes promote cell proliferation (cyclin D,
cyclin B, c-Myc), angiogenesis (VEGF, HIF1), cell survival (Bcl-2, sur-
vivin), and metastasis (MMP-1, MMP-9)40–41. Therefore, overactivation
of STAT3 results in increased expression of target genes which con-
tributes to the aggressive tumor phenotype42. Further, HCCLM3 cells
were incubated with MNK8 (25 µM) for various duration and the
expression of apoptosis-related proteins was analyzed. MNK8 decreased
the expression of antiapoptotic proteins such as Bcl-2, survivin, and
3

A.M. Sajith et al.
Bioorganic & Medicinal Chemistry Letters 50 (2021) 128332
Fig. 3. (A & B) The cell lysate prepared from MNK8
(25
μM)-treated HepG2 and HCCLM3 cells were
examined using western blotting. Time-dependent
inhibitory effects of MNK8 on the phosphorylation
of STAT3Y705 in both cell lines are shown. (C & D) The
cell lysate prepared from MNK8 (25
μM)-treated
HepG2 and HCCLM3 cells were examined using
western blotting for phosphorylation of STAT5Y694 in
both cell lines. (E & F) The cell lysate prepared from
WP1066-treated HepG2 and HCCLM3 cells was
examined using western blotting. Time-dependent
inhibitory effects of WP1066 on the phosphorylation
of STAT3Y705 in both cell lines are shown. (G) Nuclear
extract of MNK8-pretreated HepG2 and HCCLM3 cells
was prepared, and interaction between STAT3 and
DNA was analyzed (*P < 0.05; **P < 0.005).
4

A.M. Sajith et al.
Bioorganic & Medicinal Chemistry Letters 50 (2021) 128332
Fig. 4. (A) MNK8 (50
μ
M)-treated HepG2 and HCCLM3 cells were stained with propidium iodide and examined using flow cytometry. The distribution of cells in the
different phases of the cell cycle is presented as a bar graph. (B) The cell lysate prepared from MNK8 (25 μM)-treated HCCLM3 cells was examined using western
blotting. Time-dependent inhibitory effects of MNK8 on the levels of cleaved caspase 3, Bcl-2, cyclin D1, survivin, and β-actin.
cyclin D1 (Fig. 4B). Therefore, it may be interpreted that the reduction in
the expression of these proteins is due to inhibition of STAT3 activation
which subsequently results in apoptosis of treated cells. Concurrently,
MNK8 treatment increased cleaved fragment of caspase-3 (Fig. 4B)
suggesting that MNK8 triggers apoptosis to induce cell death. The results
of Sub-G1 analysis and western blotting are in agreement with each
other and provided direct evidence for the apoptogenic effect of MNK8.
MNK8 reduces CXCL12-driven cell migration: Migration and invasion
are critical events associated with cancer metastasis. STAT3 controls the
expression of metastasis-related transcription factors (such as Snail and
Twist) and therefore, inhibition of STAT3 is an ideal strategy to abrogate
the spreading of cancer. HepG2 and HCCLM3 cells were propagated in
the migration chamber until it is confluent. Thereafter, a cell-free space
of 500 µm was made by pulling out the culture-insert, and the chamber is
supplemented with MNK8 in the presence and absence of CXCL12.
MNK8 repressed the migration of cells towards the cell-free space in the
culture dish (Fig. 5A). A similar inhibitory effect was observed in the
culture flask supplemented with CXCL12 indicating the antimigratory
effect of MNK8 towards HCC cells.
for 8 h. Thereafter, the invasion chamber is placed in a plate either
containing the basal medium or basal medium supplemented with
CXCL12. The number of invaded cells towards the opposite side of the
transwell chamber was studied using crystal violet staining. MNK8
effectively mitigated the CXCL12-driven invasive potential of both cell
lines (Fig. 5B). We have previously shown that STAT3 inhibitors abro-
gate cell mobility and metastasis in in vitro and in vivo models43–45
.
Therefore, the antimigratory and anti-invasive properties of MNK8
could be due to the inhibition of the STAT3 signaling pathway.
In conclusion, a synthetic pyrimidine-2,4-dione prepared using ionic
liquids has been demonstrated to induce anticancer activity via inhib-
iting the STAT3 signaling pathway in HCC cells. This compound may be
used to evaluate its cytotoxic potential in other STAT3-positive cancers.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
MNK8 reduces CXCL12-driven cell invasion: HepG2 and HCCLM3 cells
were grown in a medium devoid of serum in the upper chamber of the
invasion system and pre-incubated in the presence or absence of MNK8
5

A.M. Sajith et al.
Bioorganic & Medicinal Chemistry Letters 50 (2021) 128332
Fig. 5. (A) HepG2 and HCCLM3 cells were propagated in the migration chamber, cell-free space is created and incubated with MNK8 either with or without CXCL12
(100 ng/mL) as described in materials and methods. The width of the cell-free space was measured before and after treatment with MNK8 alone, CXCL12 alone, and
MNK8 + CXCL12. Error bars, S.E. (B) HepG2 and HCCLM3 cells were seeded into an invasion chamber in a serum-free medium, followed by transferring into a 24-
well plate and treatment with MNK8 either with or without CXCL12 (100 ng/mL) as described materials and methods. At last, the chamber was examined for
visualization of invaded cells. Error bars, S.E.
[6] Lee JH, Chiang SY, Nam D, et al. Capillarisin inhibits constitutive and inducible
Acknowledgments
STAT3 activation through induction of SHP-1 and SHP-2 tyrosine phosphatases.
Cancer Lett. 2014;345:140–148.
K.S.R. thanks the University Grants Commission, New Delhi, and
Council of Scientific & Industrial Research for providing the Basic Sci-
ence Research faculty fellowship and Emeritus Scientist, respectively. K.
S.R., and C.D.M., thanks DST-Promotion of University Research and
Scientific (PURSE), Institution of Excellence, University of Mysore for
providing laboratory facility. This project was supported by Researchers
Supporting Project number (RSP-2021/383) King Saud University,
Riyadh, Saudi Arabia.
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Appendix A. Supplementary data
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of hepatocellular carcinoma cells through targeting STAT3 signaling pathway.
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Supplementary data to this article can be found online at https://doi.
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