Journal of Pharmacological Sciences 148 (2022) 93e102

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Journal of Pharmacological Sciences

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Effect of Piper cubeba total extract and isolated lignans on head and
neck cancer cell lines and normal fibroblasts
Juliana Prado Gusson-Zanetoni a, Julliene Stephanie Guaraldi Monteiro da Silva a,
~o a, Laila Toniol Cardin a, Janesly Prates a, Stefanie Oliveira Sousa a,
Thais Bravo Pica
Tiago Henrique b, Sonia Maria Oliani a, Eloiza Helena Tajara b, c,
Marcio Luis Andrade e Silva d, Nayanne Larissa Cunha d, Ana Carolina da Silva Gomes e,
via Cristina Rodrigues-Lisoni a, f, *
Rosangela Silva Laurentiz e, Fla
a ~ ~o Jos
Sao Paulo State University (UNESP), Institute of Biosciences, Humanities and Exact Science (IBILCE), Campus Sa e do Rio Preto, Department of Biology,
SP, Brazil
b ~o Jos
School of Medicine of Sa e do Rio Preto (FAMERP), Department of Molecular Biology, Sa ~o Jos
e do Rio Preto, Brazil
c ~o Paulo, Sa
Department of Genetics and Evolutive Biology, Institute of Biosciences, University of Sa ~o Paulo, SP, Brazil
d
University of Franca (UNIFRAN), Nucleus of Research in Exact and Technological Sciences, Franca, SP, Brazil
e ~
Sao Paulo State University (UNESP), School of Engineering (FEIS), Campus Ilha Solteira, Department of Physical Chemistry, Brazil
f ~
Sao Paulo State University (UNESP), School of Engineering (FEIS), Campus Ilha Solteira, Department of Biology and Animal Science, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The objective of the present study was to evaluate the action of the crude hydroalcoholic extract of Piper
Received 13 August 2020 cubeba fruits and isolated lignans (cubebin, dihydrocubebin, ethylcubebin, hinokinin and methylcubebin)
Received in revised form on head and neck cancer cells. We evaluated the influence of the Piper cubeba extract and isolated lignans
1 September 2021
(10, 50 e 100 mg/mL) for 4, 24, 48 and 72 h, in the larynx (Hep-2) and oral (SCC-25) squamous cell
Accepted 6 September 2021
carcinoma cells and normal fibroblasts, on morphology, cell proliferation and migration, cytotoxicity,
Available online 7 October 2021
genotoxicity and gene and protein expression (PTGS2, PTGER3, PTGER4, MMP2, MMP9). The results
showed that the P. cubeba extract and different lignans do not alter the cellular morphology, but decrease
Keywords:
Phytotherapy
cell proliferation and migration, have low cytotoxic and genotoxic effects, probably due to the alteration
Bioactive compounds of the expression of genes and proteins involved with inflammatory process. From these data, we can
Anti-inflammatory agents conclude that the lignans cubebin and methylcubebin had a greater effect on head and neck cancer cells
in the antiproliferative, antimigratory and genotoxic action, and could be the target of the development
of new therapies including possible new drugs as a therapeutic resource for the treatment of head and
neck cancer due to its immense range of biological properties.
© 2021 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological
Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/
4.0/).

1. Introduction oral cavity, oropharynx, hypopharynx and larynx, accounting for

Head and neck cancer is the sixth most common cancer diag-
nosis worldwide. This type of cancer comprises a group of ma-
lignant diseases that affect the mucosal lining in different
anatomical sites, including the nasopharynx, paranasal sinuses,
* Corresponding author. Department of Biology and Animal Science, S~
State University (Unesp), School of Engineering, Av. Brazil, 56, CEP: 15385-000 Ilha
~o Paulo, Brazil.
Solteira, Sa
E-mail address: flavia.lisoni@unesp.br (F.C. Rodrigues-Lisoni).
Peer review under responsibility of Japanese Pharmacological Society.
more than 650,000 new cases of cancer annually and more of
350,000 deaths.1
The main risk factors for the development of HNSCC are alcohol
consumption, smoking, and infection with the human papilloma-
virus (HPV).2 Surgery, including open approaches, endoscopic ap-
proaches and robotic surgery, is considered the standard of care for
most oral cavity cancers. Radiotherapy and simultaneous chemo-
radiation are used for early and advanced cancers of other regions
ao Paulo
of the head and neck. Chemotherapy with epidermal growth factor
receptor inhibitor and immune checkpoint inhibitors are now
considered the standard of care for head and neck cancer.3

https://doi.org/10.1016/j.jphs.2021.09.002
1347-8613/© 2021 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
 ~o et al.
J.P. Gusson-Zanetoni, J.S.G.M. da Silva, T.B. Pica
In addition to these interventions already known for the treat-
ment of cancer, some plants stand out in popular medicine have
aroused the interest of scientists. An example of one of these is
Piper cubeba, popularly known as Java pepper, which is extensively
used in traditional medicine in Indonesia.4 The bioactive com-
pounds of Piper cubeba have been of great value in the discovery of
new therapeutic agents to treat diseases associated with inflam-
matory problems, renal disorders, syphilis, abdominal pain, enter-
itis and asthma.5
The biological properties of P. cubeba are directly related to
the presence of lignans such as cubebin, hinokinin, dihy-
drocubebin among others, already studied by different groups
also in other plants.6 Lignans are special metabolites produced by
plants and are generally associated with plant defense against
the action of pathogens, being frequent in the Piper genus.7
Several lignans exert anti-cancer, antioxidant, antimicrobial,
anti-inflammatory, immunosuppressive and hepatoprotective
effects.8
Only few reports in the literature have associated genetic
pathways to the specific action of P. cubeba.9 One of these pathways
is mediated by cyclooxygenase 2 (PTGS2), an enzyme responsible
for the conversion of arachidonic acid to prostaglandins, prosta-
cyclins and thromboxane, which are fundamental in the process of
tissue inflammation. Increased levels of PTGS2 have been reported
in several types of cancer, for example pancreatic, lung, breast,
stomach, colon, prostate and head and neck cancers.10 PTGS2 gene
encodes prostaglandin E2 (PGE2), which can activate G protein-
coupled receptors such as PTGER1, PTGER2, PTGER3, and PTGER4,
and regulates the expression, secretion, and activation of various
metalloproteinases (MMPs) in different cell types.11
MMPs are a group of zinc-dependent endopeptidases that are
known primarily for their ability to degrade extracellular matrix
components facilitating tumor progression.12 Several MMPs have
high expression in head and neck cancer including MMP2, MMP8,
MMP9 and MMP13. MMP2 and MMP9 play an important role in head
and neck carcinogenesis by degrading type IV collagen, the main
component of the basement membrane.13
Considering the literature data, the present study was designed
to investigate the potential cytotoxic and genotoxic effect of these
compounds extracted from P. cubeba and their influence on gene
and protein expression, besides to expand the understanding of the
use of these phytotherapic as a therapeutic alternative.
2. Materials and methods
2.1. Hep-2, SCC-25 and fibroblast culture conditions
The cancer cell lines studied were derived from laryngeal (Hep-
2) and tongue (SCC-25) epidermoid carcinomas, both from the
American Type Culture Collection (ATCC). Normal human oral fi-
broblasts were kindly provided by Professor Ricardo Coletta, Uni-
versity of Campinas, Piracicaba, SP, Brazil (F5). The Hep-2 cell line
was seeded in MEM-Earle medium complete medium (Cultilab, Br),
pH 7.5, supplemented with 10% bovine serum (BSA) (Cultilab, Br),
1% antibiotic/antimycotic (Invitrogen, Carlsbad, USA, UK), 1% L-
glutamine (200 mM) (Sigma Aldrich, USA), 1% 10 mM non-essential
amino acids (Sigma Aldrich, USA) and 1% 100 mM sodium pyru-
vate (Sigma Aldrich, USA). The SCC-25 cell line was seed in DMEM-
HAM-F12 medium (Cultilab, Br) and fibroblasts in DMEM high
glucose medium (Cultilab, Br), both supplemented with the Hep-
2 cell line. These cells were maintained at 37
C and 5% CO2
atmosphere.
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Journal of Pharmacological Sciences 148 (2022) 93e102
2.2. Plant materials and isolation of major compounds
P. cubeba fruits were macerated with 70% aqueous ethanol
(500 g seed for 2 L ethanol) for five days. The extract was filtered
and concentrated under vacuum to obtain the crude hydroalcoholic
extract, which was fractionated in hexane and methanol/water
phases (9:1).7 The crude methanol/water fraction (50 g) was sub-
mitted to three sequential silica gel columns chromatography using
gradient crescent of the mixture of hexane/ethyl acetate yielding
five fractions with similar profiles. These fractions were then
combined and again submitted (separated) to new silica gel column
to obtain five major compounds (cubebin, dihydrocubebin, ethyl-
cubebin, hinokinin and methylcubebin). The structure of these
compounds was determined using 1H and 13C NMR (supplementary
data).
2.3. Pharmacological treatment
The total extract and the lignan extracts were diluted in 0.3%
DMSO (dimethylsulfoxide e DMSO, Sigma Aldrich, USA). Podo-
phyllotoxin (Sigma Aldrich, USA), a lignan with proven cytotoxic
activity, was also diluted in DMSO and used as a positive control14
whereas DMSO was used as a negative control. Lignans were added
to cell cultures at concentrations of 10, 50 and 100 m/mL (deter-
mined by previous studies).
2.4. Proliferation assay and cellular morphology analysis
Cells were seeded in triplicate in 6-well culture plates at a
density of 5  104 in 2 mL culture. After 24 h, the medium was
replaced with a serum-free medium for cell synchronization. After
a further 24 h, the medium was replaced again with complete
medium supplemented with lignan extracts according to the
experimental groups. Cells were analyzed and counted at four
different times (4, 24, 48, 72 h). The cell morphology was evaluated
under inverted microscopy using an Olympus CKX41.
2.5. Viability/cytotoxicity assay (MTS)
Cytotoxicity was determined by MTS assay according to the
protocol of CellTiter 96®Aquee One Solution Cell Proliferation
Assay (Promega, USA). Briefly, Hep-2, SCC-25 and fibroblasts cells
were seeded at a density of 5  103/well and exposed to lignan
extracts in triplicates. Following incubation of the plates by 4, 24, 48
or 72 h at 37
C, 20 ml of MTS reagent was added to each well.
Cytotoxicity was determined by measuring the optical density at
492 nm using an ELISA plate reader. The 50% inhibitory concen-
tration (IC50) was determined for each lignan extract treatment
and compared to controls.
2.6. Transwell cell migration assay
Cells were cultured for 24 h in 24-well plates (density of 5  104)
in a serum-free medium for cell synchronization medium for cell
synchronization, and treated with lignan extracts in triplicates. The
cells were then trypsinized and reseeded at a density of 3  104/
well on the Transwell inserts (8 mm in pore diameter, Corning
Costar, New York, USA). Cell suspension in 150 mL of serum-free
medium was added to the Transwell upper chamber and cells in
500 mL of complete medium were added to the lower chamber. The
plate was incubated at 37
C in a humidified CO2 incubator for 24 h.
The cells that migrated to the lower surface of the filter were fixed
 ~o et al.
J.P. Gusson-Zanetoni, J.S.G.M. da Silva, T.B. Pica
with 4% paraformaldehyde in PBS for 30 min and stained with
Crystal Violet solution for 30 min. The inserts were then photo-
graphed in five random fields.
2.7. Comet assay
Hep-2, SCC-25 and Fibroblasts cells were grown and treated
with lignan extracts in 12-well plates at 3  104/well in complete
medium, then the cells were trypsinized, centrifuged and the pellet
was suspended in low-melting-point (LMP) agarose and placed
onto slides precoated with normal-melting-point agarose (NMP).
The slides were placed in phosphate buffer solution (PBS), followed
by the lysis step in pH10 lysis solution (2.5 M NaCl, 100 mM EDTA,
10 mM Tris, 35 mM Lauril) at 4
C for 60 min. Electrophoresis was
performed for 20 min at 25 V/300 mA in a TriseEDTA buffer. The
material was neutralized, fixed, stained with a solution of Gel Red
10000x, 1 M NaCl and distilled water, and analyzed under a fluo-
rescence microscope in magnification of 400x. One hundred cells
per experimental group were analyzed. The DNA damage was
visually evaluated according to the intensity and comet tail
length.15
2.8. Quantitative real time PCR
Five genes (PTGS2, PTGER3, PTGER4, MMP2 and MMP9) were
selected from those reported in the literature to be associated with
inflammation.16 Reactions were performed in triplicate on the 7500
Fast Time Thermal PCR System (Applied Biosystems). The relative
Fig. 1. General Classes of lignans. Chemical formula of lignans isolated from Piper cubeba fruits: ethylcubebin, methylcubebin, hinokinin, cubebin and dihydrocubebin.
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Journal of Pharmacological Sciences 148 (2022) 93e102
expression levels of candidate genes were normalized to the
endogenous control (GAPDH/glyceraldehyde-3-phosphate dehy-
drogenase) and relative to the control sample by using the 2(eDDCt)
method.17
2.9. Protein expression evaluation
Hep-2 and SCC-25 cells were seeded at a density of 10  105 for
the evaluation of PTGS2 (1:500 mL Abcam, Cambridge, UK) and
MMP2 (1:500 mL, ABclonal, Woburn, USA) proteins and submitted
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE), then transferred to a nitrocellulose membrane (Immobilon
0.45 mm, Millipore) in a wet transfer apparatus (Bio-Rad).
Blocking was performed in Tris-buffered saline, containing 5%
skimmed milk powder and 0.05% Tween 20 (TBS-T). Blots were
incubated with the respective secondary antibodies (1: 2000 mL
Abcam, Cambridge, UK) diluted in (TBS-T) for 1 h at room tem-
perature. Proteins were visualized using the ECL detection system
(Bio-Rad, USA) for the detection of reactive bands by chem-
iluminescence visualized on Hyperfilm photographic film, and
densitometric analysis was performed by Image J software (NIH,
Maryland, USA). b-actin was used as an endogenous control
(1:5000 mL, Cell Signaling, Danvers, USA).
2.10. Statistical analyzes
After the cell proliferation and cytotoxicity tests, the statistical
analysis of variance for multiple comparisons (ANOVA) was
 ~o et al.
J.P. Gusson-Zanetoni, J.S.G.M. da Silva, T.B. Pica
Fig. 2. Effects of cubebin treatment on Hep-2, SCC-25 and Fibroblasts morphology.
Analysis of the morphology on Hep-2 (A and B), SCC-25 (C and D) and Fibroblast (E and
F) cells. The same morphology characters can be observed in control (A, C and E) and
cubebin (100 mg/ml) treated (B, D and F) cells. 400 magnification, 40 mm scale.
applied, followed by the Bonferroni adjustment, so the concentra-
tion of 100 mg/mL was chosen for all the lignans studied and the
72 h for the Hep-2 and SCC-25 cell lines and 24 h for fibroblasts.
The data were analyzed using Prisma®GraphPad software
version 5.00. Results were presented as mean ± standard error of
the mean (SEM). Values of p  0.05 were considered to be statis-
tically significant.
3. Results
3.1. Piper cubeba total extract and isolated lignans
After extraction with ethanol, 500 g of the powered P. cubeba
fruit furnished 78 g of crude ethanolic extract (15% yield).
Sequential silica gel columns chromatography produced the lignans
ethylcubebin (0.030 g), methylcubebin (0.022 g), hinokinin
(0.080 g), cubebin (0.072 g) and dihydrocubebin (0.060 g). Results
of the NMR analysis (supplementary data) confirmed the structures
of these compounds (Fig. 1). The NMR data obtained for these
compounds are in agreement with literature data.
3.2. Piper cubeba total extract and isolated lignans decrease
proliferation without modifying cellular morphology
The Hep-2, SCC-25 and fibroblasts cells did not change their
morphology characters, after the treatment with all the lignans,
total extract and podophyllotoxin, when compared to the control
group (Fig. 2). But, in the proliferation assay, all the compounds
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Journal of Pharmacological Sciences 148 (2022) 93e102
reduced cell growth in Hep-2 cells, in the concentrations of 50 and
100 mg/mL at 4, 24, 48 and 72 h (Fig. 3A). This effect of the treat-
ments on Hep-2 cells, in which there was a decrease in cell pro-
liferation in the first hours, may have occurred because the cells
had suffered some stress, for example due to trypsinization and,
therefore, with the application of the treatment, they responded to
this effect quickly.
For the SCC-25 cells, we observe that all the lignans decreased
the cell growth, in the concentration of 100 mg/mL at 72 h and the
total extract also reduced the cell growth, in the concentrations of
10 and 100 mg/mL at 48 h (Fig. 3B). However, for the fibroblasts, all
the lignans, total extract and podophyllotoxin reduced cell growth,
in the concentration of 100 mg/mL at 24 and 48 h (Fig. 3C).
3.3. Piper cubeba total extract and isolated lignans have low
cytotoxicity
For the Hep-2 cell line, when treated with the lignans (cubebin,
methylcubebin, ethylcubebin, dihydrocubebin), total extract and
podophyllotoxin presented a greater viability, compared to the
control group, in concentrations of 10, 50 and 100 mg/mL, being
more prevalent at 4 and 72 h (Fig. 4A). On the other hand, the cells
treated with lignan hinokinin showed lower cytotoxicity, in the
concentration of 100 mg/mL at 48 h, when compared to the control.
In the SCC-25 cell line, we observed that the lignans (cubebin
and dihydrocubebin), total extract and podophyllotoxin reduced
cytotoxicity, mainly in concentrations of 10 and 50 mg/mL, after
72 h. For the lignan ethylcubebin, any concentration studied
modified the cytotoxicity, in relation to the control, in the times
analyzed (Fig. 4B).
For the fibroblasts, the cytotoxicity was lower in relation to the
control when treated with cubebin in the concentrations of 10 and
50 mg/mL at 48 h, being that in the concentration of 10 mg/mL this
lignan was cytotoxic, because its IC50 was less than 50%. Fibroblasts
treated with methylcubebin, dihydrocubebin and hinokinin had a
decrease in cytotoxicity, in relation to the control, at 48 and 72 h,
but without being cytotoxic, since IC50 was greater than 50%
(Fig. 4C).
3.4. Piper cubeba total extract and isolated lignans decrease cell
migration
In the Hep-2 cell line, treatments with lignans (cubebin, dihy-
drocubebin, ethylcubebin, hinokinin), total extract and podo-
phyllotoxin decreased the migration of these cells in relation to the
control, statistically. In fact, the podophyllotoxin, used as a positive
control, showed the greatest decrease in cell migration. Only the
methylcubebin does not present significant statistical difference
(Fig. 5A).
For the SCC-25 cell line, a significant decrease in cell migration
after treatment with the lignans dihydrocubebin, ethylcubebin,
methylcubebin and podophyllotoxin was observed in relation to
the control, based on the statistical test. We can also observe that
cubebin, hinokinin and total extract reduced the cellular migration
rate, but did not present significant difference in relation to the
control (Fig. 5B).
In the fibroblasts, it was observed that only the lignan ethyl-
cubebin exerted a smaller migratory effect when compared to the
control statistically. However, cubebin, dihydrocubebin, methyl-
cubebin and podophyllotoxin showed a higher number of migrated
cells than the control, probably these lignans have a low action on
cell migration in the normal cells. The hinokinin and total extract
 ~o et al.
J.P. Gusson-Zanetoni, J.S.G.M. da Silva, T.B. Pica Journal of Pharmacological Sciences 148 (2022) 93e102

Fig. 3. Effects of lignans treatments on cell proliferation. Quantification of Hep-2 (A), SCC-25 (B) and Fibroblast (C) cells at the time of 4, 24, 48 and 72 h in the control (DMSO)
and after treatment with compounds extracted from Piper cubeba at three concentrations (10, 50 and 100 mg/ml). Symbol: * vs control; value p < 0.05.

decreased the migration rate, but did not present significant dif-
ference in relation to the control (Fig. 5C).
3.5. Damage to the genetic material of the cells can be caused by
the action of P. cubeba total extract and isolated lignans
For the Hep-2 cell line, we can observe that cubebin and
podophyllotoxin presented higher significant indices of damage in
their nuclei than the control cells (Fig. 5D). In relation to the SCC-
25 cell line, all lignans, total extract and podophyllotoxin caused
significant damage to the nuclei of these treated cells compared to
the control (Fig. 5E).
In the fibroblasts, only cubebin and methylcubebin generated
more lesions to the DNA, statistically, than in the control cells. The
damage index of the cells treated with others compounds were
equivalent to the damage of the control cells (Fig. 5F).

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J.P. Gusson-Zanetoni, J.S.G.M. da Silva, T.B. Pica Journal of Pharmacological Sciences 148 (2022) 93e102

Fig. 4. Effects of lignans treatments on cytotoxicity. Effects of the treatments with the Piper cubeba lignans for the colorimetric cell viability/cytotoxicity (MTS) method in the
Hep-2 (A), SCC-25 (B) and Fibroblast (C) cell lines. Graph with axis X ¼ time (hours) and y ¼ viability (percentage). Symbol: * vs control; value p < 0.05.

3.6. Piper cubeba total extract and isolated lignans alter gene
expression
The Hep-2 cell line (Fig. 6A), when treated with the different
lignans, total extract and podophyllotoxin showed increased
expression of the PTGER3, PTGER4 and MMP9 genes and decreased
PTGS2 and MMP2 genes compared to the untreated control group.
The SCC-25 cell line (Fig. 6B), when treated with the different
compounds, showed increased expression of PTGS2 and MMP9,
genes and decreased PTGER3, PTGER4 and MMP2 genes compared to
the untreated control group. Normal oral fibroblasts (Fig. 6C), when
98
treated with the different lignans, showed increased expression,
generally, of the PTGS2, PTGER3, MMP2 and MMP9 genes and
decreased PTGER4 gene compared to the untreated control group.
3.7. Lignans from P. cubeba alter the expression of PTGS2 and
MMP2 proteins in head and neck cancer cells
The expression of PTGS2 and MMP2 proteins were examined
using the Western blot technique to evaluate the effects of cubebin
and methylcubebin lignans, as well as podophyllotoxin, as these
compounds had greater effects on head and neck cancer cells in
 ~o et al.
J.P. Gusson-Zanetoni, J.S.G.M. da Silva, T.B. Pica Journal of Pharmacological Sciences 148 (2022) 93e102

Fig. 5. Effects of lignans treatments on cell migration and genotoxicity. Transwell migration assay of Hep-2 (A), SCC-25 (B) and Fibroblast (C) cells with densitometry repre-
sentation of the number of migrated cells. Genotoxicity assay of Hep-2 (D), SCC-25 (E) and Fibroblast (F) cells treated with total extract and lignans extracted from Piper cubeba
(cubebin, dihydrocubebin, ethylcubebin, hinokinin, methylcubebin and podophyllotoxin) at 72 h for Hep-2 and SCC-25 and 24 h for fibroblasts at the concentration of 100 mg/mL.
Symbol: * vs control; value p < 0.05.

previous assays (Fig. 7). The results show that in the Hep-2 cell line,
treatment with cubebin and podophyllotoxin can significantly
decrease the levels of PTGS2 protein compared to the control.
Regarding the SCC-25 cell line, treatment with lignan methyl-
cubebin significantly reduced the expression of PTGS2 protein
when compared to the untreated control. These data suggest that
lignans modify the expression of PTGS2 protein levels in head and
neck tumorigenic cells. Regarding the MMP2 protein, cubebin lig-
nan also significantly decreased the expression of this protein in the
SCC-25 lineage. As for the Hep-2 cell line, we observed a higher
expression of MMP2 protein from treatment with the lignans
cubebin and methylcubebin, and for the treatment with podo-
phyllotoxin we found that there was a decrease in the protein
expression of MMP2, but without being statistically significant in
relation to control.
4. Discussion
In the present work, we observed that the morphology of Hep-2,
SCC-25 and fibroblasts cells did not change after the treatments
with total extract and isolated lignans from P. cubeba fruits. Despite
this, we observed a decrease in cell proliferation after the

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 ~o et al.
J.P. Gusson-Zanetoni, J.S.G.M. da Silva, T.B. Pica Journal of Pharmacological Sciences 148 (2022) 93e102

Fig. 6. Modulation of mRNA expression by lignans treatments. Analysis of PTGS2, PTGER3, PTGER4, MMP2 and MMP9 expression by real-time PCR for the Hep-2 (A), SCC-25 (B)
and fibroblast cells (C) performed in triplicates. mRNA expression was considered significant exhibiting values  1.0 or  1.0 versus control, based on logarithm 2.

Fig. 7. Effect of cubebin, methylcubebin and podophyllotoxin lignans on PTGS2 and MMP2 protein expression. Data were presented as the mean ± SEM performed from three
independent experiments. Symbol: * vs control; value p < 0.05.

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J.P. Gusson-Zanetoni, J.S.G.M. da Silva, T.B. Pica
treatments in these cells, evidencing the involvement of these
lignans in antiproliferation mechanisms, as well as Cunha and
colleagues9 also verified the antiproliferative effect of hinokinin
combined with doxorubicin in breast cancer cell lines (MCF-7 and
SKBR-3) by modulation of the cyclin-dependent kinase inhibitor 1.
The cubebin also inhibited proliferation in human prostate cancer
cells (LNCaP) by reducing DNA synthesis and inducing apoptosis,
thus this lignan has an antitumor action as a biological activity.18
In the cytotoxicity experiment, by the MTS assay, it was found
that these lignans show little cytotoxicity for the Hep-2, SCC-25 and
fibroblast cells. That is, the lignans structural differences extracted
from P. cubeba do not lead to significant differences on the cyto-
toxicity observed on the three types of cells evaluated. That it is
good, because in the tumoral microenvironment, there are normal
and tumor cells in the same place, then if the drug is not cytotoxic is
good to the patient and probably the cell's dead are not occur
because the compound is toxic, but probably this occur for another
way. Of course, if these compounds were cytotoxic to neoplastic
cells and only death of these cells would be better, but lignans were
not cytotoxic to any cells, except fibroblasts when treated with
cubebin at concentration of 10 mg/mL, which was cytotoxic due to
its IC50 being below 50%. According to Rajalekshmi et al. (2016),
cubebin and its synthetic derivatives, also showed cytotoxicity ef-
fect against different human cancer cell lines, by MTT assay.19
With respect to the cell migration assay, it was found that
lignans promoted an anti-migratory effect in the Hep-2 and SCC-25
and fibroblasts cells, in which the same was not observed by da
Silva et al.20 where the compounds derived from cubebin lignan did
not exhibit activity in the cell migration test, investigated using
different animal models such as rats and mice.
Interestingly, ethylcubebin had an anti-migratory effect even on
fibroblasts, which can be explained as it is the most hydrophobic
among the lignans evaluated, that is, originally it may be easier to
penetrate into healthy cells and with greater regularity in the shape
than the other cell lines studied.
In our genotoxicity study, some lignans caused damage to Hep-
2, SCC-25 and fibroblast nuclei. Other authors analyzing mouse
cells, observed that the cubebin also produced genotoxic effects,
investigated by micronuclei (MN) assays on bone marrow cells and
comet assay on peripheral blood lymphocytes.21 In the study by
Resende et al.4 however, it was observed that lignan hinokinin had
no genotoxic effect seen by the comet assay on Chinese hamster
lung fibroblast cell (V79), an effect also seen on Hep-2 and normal
fibroblasts cells in our study.
In order to complete our investigation of the effect of P. cubeba
extract and lignans on the inflammatory response in Hep-2, SCC-25
and fibroblast cells, we verified the expression of five genes (PTGS2,
PTGER3, PTGER4, MMP2 and MMP9) related to inflammation pro-
cesses. We also analyzed PTGS2 and MMP2 proteins by Western
blot assay for cubebin, methylcubebin and podophyllotoxin lignans,
these compounds being the ones that showed anti-tumorigenic
effects through antiproliferative, antimigratory and genotoxic ac-
tion on head and neck cancer cells. Hep-2 cell line showed the
reduction of PTGS2 gene expression after treatment with P. cubeba
extract and lignans, but in the SCC-25 and fibroblast cells, the
lignans act to increase the expression of this gene. PTGS2 is an
inducible expression enzyme responsible for the production of
prostaglandins at sites of inflammation, playing a role in the initi-
ation and progression of various human diseases.22 In addition, the
PTGS2 gene is often overexpressed in a variety of human neoplasms,
including laryngeal carcinoma.23
Thus, our findings with the Hep-2 laryngeal neoplastic lineage
show that treatment with compounds extracted from P. cubeba can
regulate PTGS2 expression, making it less expressed. In this sense
and as expected, our Western blot results revealed that treatment
101
Journal of Pharmacological Sciences 148 (2022) 93e102
with cubebin significantly decreases the level of PTGS2 protein
expression in Hep-2 laryngeal cancer cells. In previous studies it was
seen that cubebin can act by inhibiting cyclooxygenase-2 (PTGS2) in
rat paw edema using prostaglandin as a mediator.24 This decrease in
PTGS2 protein expression was also observed when treatment with
methylcubebin was performed in SCC-25 tongue cells. Thus, cubebin
and methylcubebin lignans decrease PTGS2 protein expression in
head and neck cancer cells in the present study.
The PTGER3 gene presented low expression after the lignans
treatments and total extract in the SCC-25 cells, differently in Hep-2
and fibroblast cells, in which this gene was up regulated. These
differences in expression of the PTGER3 gene for the cells studied
may be partially related to the isoforms of this gene, since these
mainly signal the Gi (inhibitory) proteins, but also, partially, by the
Ca2þ phospholipase cascade or the Gs (stimulatory) proteins.25
As expected, the PTGER4 gene showed low expression in SCC-25
and fibroblast cells after the treatments. In the Hep-2 cell line, the
expression of this gene was up regulated. In this way, the inacti-
vation or negative expression of the PTGER4 receptor promotes
tumorigenic reduction effects, and may be caused by blocking the
survival of PGE2-dependent tumor cells, or due to a reduction in
the blood supply necessary for oxygenation, proliferation and sur-
vival of tumor cells.26 Studies on the roles of PTGS2 receptors and
PTGERs in head and neck carcinomas have revealed a significant
increase in PTGS2 expression as well as all PTGER receptors,27 but
when the treatments (dihydrocubebin, total extract, hinokinin,
methylcubebin and podophyllotoxin) reduce the PTGER4 expres-
sion means that the treatment is acting. Then, we can suppose that
these lignans could be effective in the treatment of the tongue
squamous cell carcinoma, like in the SCC-25 cells, but not so
effective in larynx squamous cell carcinoma, like Hep-2 cells.
Regarding matrix metalloproteinases, we found a lower expres-
sion of MMP2 gene in Hep-2 and SCC-25 cell lines, and a greater
expression in fibroblasts. The expression level of MMP2 protein was
also reduced in SCC-25 treated with cubebin, methylcubebin and
podophyllotoxin, seen in the Western blot assay. This diminished
MMP2 gene and protein expression in the SCC-25 tumorigenic cell
line may be related to our results of the antimigratory effect of the
lignans and total extract studied, because it is known that different
groups of proteolytic enzymes are involved in the matrix breakdown
and among them, the most important group in tumor invasion and
metastasis are the genes MMPs.28 Even as in our research, the lignans
and total extract of P. cubeba down regulated MMP2 and MMP9 gene
expression and had an antimigratory effect.
For metalloproteinase MMP9, an increase in its expression was
observed in all cells analyzed. In contrast to the study by Mali
et al.29 in which they investigated the action of enterolactone, they
found that it significantly reduced the expression of the MMP2 and
MMP9 genes in MCF-7 and MDA MB 231 breast cancer cells.
Probably not all the metalloproteinases undergo the effects exerted
by the lignans in the cells studied by our group.
The lignans extracted from P. cubeba have structural differences
only in relation to the 4-membered ring, where in cubebin this ring
is a lactol ring, in hynokinin is a lactone, in methylcubebin and
ethylcubebin is a ketal, while in dihydrocubebin there is a linked
CH2OH group to the carbons where the ring binds to the other
lignans (Fig. 1). These structural differences alter the degree of
solubility, lipophilicity, greater or lesser rigidity of the molecule and
affect the way these lignans interact in the cells, leading to differ-
ences or similarities in their biological properties as observed. This
4-membered ring, or the absence of it in dihydrocubebin, makes
the molecules assume different conformations that can have
different models of interaction with the proteins that act in these
metabolic pathways, inhibiting or enhancing the expression of the
studied genes.
 ~o et al.
J.P. Gusson-Zanetoni, J.S.G.M. da Silva, T.B. Pica
5. Conclusion
From these data, we can conclude that the different lignans do
not alter the cellular morphology, but they diminish the prolifera-
tion and migration of the cells, being little cytotoxic, but with
genotoxic effects, probably for modulating the expression of genes
and proteins related to the inflammatory process. We also observed
that the lignans cubebin and methylcubebin had a greater effect on
head and neck cancer cells in the antiproliferative, antimigratory
and genotoxic action, and could be the target of the development of
new therapies for the treatment of head and neck cancer.
Author contributions
Juliana Prado Gusson Zanetoni: Conceptualization, Methodol-
ogy, Analysis, Research, Writing. Julliene Stephanie Guaraldi Mon-
teiro da Silva: Methodology. Thais Bravo Pica ~o: Methodology. Laila
Toniol Cardin: Methodology. Janesly Prates: Methodology. Stefanie
Oliveira Sousa: Methodology. Tiago Henrique: Methodology and
Review. Sonia Maria Oliani: Appeal and Review. Eloiza Helena
Tajara: Review and Editing. Marcio Luis Andrade e Silva: Method-
ology. Nayane Larissa Cunha: Methodology. Ana Carolina da Silva
Gomes: Methodology. Rosangela Silva Laurentiz: Resource, Review
and Editing. Flavia Cristina Rodrigues-Lisoni: Conceptualization,
Writing, Review, Editing, Supervision and Resources.
Funding
This work was supported by the Foundation for Research Sup-
~o Paulo - FAPESP (2017/02100e3 to FCR-L);
port of the State of Sa
Coordination of Improvement of Higher Level Personnel - CAPES for
the JPG scholarship.
Declaration of competing interest
The authors declared that they have no conflict of interest.
Acknowledgments
The authors thank Prof. Dr. Ricardo Coletta, University of Cam-
pinas/UNICAMP, Piracicaba, SP, for providing primary cells derived
from normal human oral fibroblasts (F5).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jphs.2021.09.002.
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