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

more than 650,000 new cases of cancer annually and more of
Head and neck cancer is the sixth most common cancer diag- 350,000 deaths.1
nosis worldwide. This type of cancer comprises a group of ma- The main risk factors for the development of HNSCC are alcohol
lignant diseases that affect the mucosal lining in different consumption, smoking, and infection with the human papilloma-
anatomical sites, including the nasopharynx, paranasal sinuses, 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
* Corresponding author. Department of Biology and Animal Science, S~ ao Paulo
State University (Unesp), School of Engineering, Av. Brazil, 56, CEP: 15385-000 Ilha of the head and neck. Chemotherapy with epidermal growth factor
~o Paulo, Brazil.
Solteira, Sa receptor inhibitor and immune checkpoint inhibitors are now
E-mail address: fl[email protected] (F.C. Rodrigues-Lisoni). considered the standard of care for head and neck cancer.3
Peer review under responsibility of Japanese Pharmacological Society.

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 Journal of Pharmacological Sciences 148 (2022) 93e102

In addition to these interventions already known for the treat- 2.2. Plant materials and isolation of major compounds
ment of cancer, some plants stand out in popular medicine have
aroused the interest of scientists. An example of one of these is P. cubeba fruits were macerated with 70% aqueous ethanol
Piper cubeba, popularly known as Java pepper, which is extensively (500 g seed for 2 L ethanol) for five days. The extract was filtered
used in traditional medicine in Indonesia.4 The bioactive com- and concentrated under vacuum to obtain the crude hydroalcoholic
pounds of Piper cubeba have been of great value in the discovery of extract, which was fractionated in hexane and methanol/water
new therapeutic agents to treat diseases associated with inflam- phases (9:1).7 The crude methanol/water fraction (50 g) was sub-
matory problems, renal disorders, syphilis, abdominal pain, enter- mitted to three sequential silica gel columns chromatography using
itis and asthma.5 gradient crescent of the mixture of hexane/ethyl acetate yielding
The biological properties of P. cubeba are directly related to five fractions with similar profiles. These fractions were then
the presence of lignans such as cubebin, hinokinin, dihy- combined and again submitted (separated) to new silica gel column
drocubebin among others, already studied by different groups to obtain five major compounds (cubebin, dihydrocubebin, ethyl-
also in other plants.6 Lignans are special metabolites produced by cubebin, hinokinin and methylcubebin). The structure of these
plants and are generally associated with plant defense against compounds was determined using 1H and 13C NMR (supplementary
the action of pathogens, being frequent in the Piper genus.7 data).
Several lignans exert anti-cancer, antioxidant, antimicrobial,
anti-inflammatory, immunosuppressive and hepatoprotective 2.3. Pharmacological treatment
effects.8
Only few reports in the literature have associated genetic The total extract and the lignan extracts were diluted in 0.3%
pathways to the specific action of P. cubeba.9 One of these pathways DMSO (dimethylsulfoxide e DMSO, Sigma Aldrich, USA). Podo-
is mediated by cyclooxygenase 2 (PTGS2), an enzyme responsible phyllotoxin (Sigma Aldrich, USA), a lignan with proven cytotoxic
for the conversion of arachidonic acid to prostaglandins, prosta- activity, was also diluted in DMSO and used as a positive control14
cyclins and thromboxane, which are fundamental in the process of whereas DMSO was used as a negative control. Lignans were added
tissue inflammation. Increased levels of PTGS2 have been reported to cell cultures at concentrations of 10, 50 and 100 m/mL (deter-
in several types of cancer, for example pancreatic, lung, breast, mined by previous studies).
stomach, colon, prostate and head and neck cancers.10 PTGS2 gene
encodes prostaglandin E2 (PGE2), which can activate G protein- 2.4. Proliferation assay and cellular morphology analysis
coupled receptors such as PTGER1, PTGER2, PTGER3, and PTGER4,
and regulates the expression, secretion, and activation of various Cells were seeded in triplicate in 6-well culture plates at a
metalloproteinases (MMPs) in different cell types.11 density of 5  104 in 2 mL culture. After 24 h, the medium was
MMPs are a group of zinc-dependent endopeptidases that are replaced with a serum-free medium for cell synchronization. After
known primarily for their ability to degrade extracellular matrix a further 24 h, the medium was replaced again with complete
components facilitating tumor progression.12 Several MMPs have medium supplemented with lignan extracts according to the
high expression in head and neck cancer including MMP2, MMP8, experimental groups. Cells were analyzed and counted at four
MMP9 and MMP13. MMP2 and MMP9 play an important role in head different times (4, 24, 48, 72 h). The cell morphology was evaluated
and neck carcinogenesis by degrading type IV collagen, the main under inverted microscopy using an Olympus CKX41.
component of the basement membrane.13
Considering the literature data, the present study was designed 2.5. Viability/cytotoxicity assay (MTS)
to investigate the potential cytotoxic and genotoxic effect of these
compounds extracted from P. cubeba and their influence on gene Cytotoxicity was determined by MTS assay according to the
and protein expression, besides to expand the understanding of the protocol of CellTiter 96®Aquee One Solution Cell Proliferation
use of these phytotherapic as a therapeutic alternative. Assay (Promega, USA). Briefly, Hep-2, SCC-25 and fibroblasts cells
were seeded at a density of 5  103/well and exposed to lignan
2. Materials and methods 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.
2.1. Hep-2, SCC-25 and fibroblast culture conditions Cytotoxicity was determined by measuring the optical density at
492 nm using an ELISA plate reader. The 50% inhibitory concen-
The cancer cell lines studied were derived from laryngeal (Hep- tration (IC50) was determined for each lignan extract treatment
2) and tongue (SCC-25) epidermoid carcinomas, both from the and compared to controls.
American Type Culture Collection (ATCC). Normal human oral fi-
broblasts were kindly provided by Professor Ricardo Coletta, Uni- 2.6. Transwell cell migration assay
versity of Campinas, Piracicaba, SP, Brazil (F5). The Hep-2 cell line
was seeded in MEM-Earle medium complete medium (Cultilab, Br), Cells were cultured for 24 h in 24-well plates (density of 5  104)
pH 7.5, supplemented with 10% bovine serum (BSA) (Cultilab, Br), in a serum-free medium for cell synchronization medium for cell
1% antibiotic/antimycotic (Invitrogen, Carlsbad, USA, UK), 1% L- synchronization, and treated with lignan extracts in triplicates. The
glutamine (200 mM) (Sigma Aldrich, USA), 1% 10 mM non-essential cells were then trypsinized and reseeded at a density of 3  104/
amino acids (Sigma Aldrich, USA) and 1% 100 mM sodium pyru- well on the Transwell inserts (8 mm in pore diameter, Corning
vate (Sigma Aldrich, USA). The SCC-25 cell line was seed in DMEM- Costar, New York, USA). Cell suspension in 150 mL of serum-free
HAM-F12 medium (Cultilab, Br) and fibroblasts in DMEM high medium was added to the Transwell upper chamber and cells in
glucose medium (Cultilab, Br), both supplemented with the Hep- 500 mL of complete medium were added to the lower chamber. The
2 cell line. These cells were maintained at 37
C and 5% CO2 plate was incubated at 37
C in a humidified CO2 incubator for 24 h.
atmosphere. The cells that migrated to the lower surface of the filter were fixed

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

with 4% paraformaldehyde in PBS for 30 min and stained with expression levels of candidate genes were normalized to the
Crystal Violet solution for 30 min. The inserts were then photo- endogenous control (GAPDH/glyceraldehyde-3-phosphate dehy-
graphed in five random fields. drogenase) and relative to the control sample by using the 2(eDDCt)
method.17
2.7. Comet assay
2.9. Protein expression evaluation
Hep-2, SCC-25 and Fibroblasts cells were grown and treated
with lignan extracts in 12-well plates at 3  104/well in complete Hep-2 and SCC-25 cells were seeded at a density of 10  105 for
medium, then the cells were trypsinized, centrifuged and the pellet the evaluation of PTGS2 (1:500 mL Abcam, Cambridge, UK) and
was suspended in low-melting-point (LMP) agarose and placed MMP2 (1:500 mL, ABclonal, Woburn, USA) proteins and submitted
onto slides precoated with normal-melting-point agarose (NMP). sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
The slides were placed in phosphate buffer solution (PBS), followed PAGE), then transferred to a nitrocellulose membrane (Immobilon
by the lysis step in pH10 lysis solution (2.5 M NaCl, 100 mM EDTA, 0.45 mm, Millipore) in a wet transfer apparatus (Bio-Rad).
10 mM Tris, 35 mM Lauril) at 4
C for 60 min. Electrophoresis was Blocking was performed in Tris-buffered saline, containing 5%
performed for 20 min at 25 V/300 mA in a TriseEDTA buffer. The skimmed milk powder and 0.05% Tween 20 (TBS-T). Blots were
material was neutralized, fixed, stained with a solution of Gel Red incubated with the respective secondary antibodies (1: 2000 mL
10000x, 1 M NaCl and distilled water, and analyzed under a fluo- Abcam, Cambridge, UK) diluted in (TBS-T) for 1 h at room tem-
rescence microscope in magnification of 400x. One hundred cells perature. Proteins were visualized using the ECL detection system
per experimental group were analyzed. The DNA damage was (Bio-Rad, USA) for the detection of reactive bands by chem-
visually evaluated according to the intensity and comet tail iluminescence visualized on Hyperfilm photographic film, and
length.15 densitometric analysis was performed by Image J software (NIH,
Maryland, USA). b-actin was used as an endogenous control
2.8. Quantitative real time PCR (1:5000 mL, Cell Signaling, Danvers, USA).

Five genes (PTGS2, PTGER3, PTGER4, MMP2 and MMP9) were 2.10. Statistical analyzes
selected from those reported in the literature to be associated with
inflammation.16 Reactions were performed in triplicate on the 7500 After the cell proliferation and cytotoxicity tests, the statistical
Fast Time Thermal PCR System (Applied Biosystems). The relative analysis of variance for multiple comparisons (ANOVA) was

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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J.P. Gusson-Zanetoni, J.S.G.M. da Silva, T.B. Pica 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
Fig. 2. Effects of cubebin treatment on Hep-2, SCC-25 and Fibroblasts morphology. modified the cytotoxicity, in relation to the control, in the times
Analysis of the morphology on Hep-2 (A and B), SCC-25 (C and D) and Fibroblast (E and analyzed (Fig. 4B).
F) cells. The same morphology characters can be observed in control (A, C and E) and
For the fibroblasts, the cytotoxicity was lower in relation to the
cubebin (100 mg/ml) treated (B, D and F) cells. 400 magnification, 40 mm scale.
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
applied, followed by the Bonferroni adjustment, so the concentra-
treated with methylcubebin, dihydrocubebin and hinokinin had a
tion of 100 mg/mL was chosen for all the lignans studied and the
decrease in cytotoxicity, in relation to the control, at 48 and 72 h,
72 h for the Hep-2 and SCC-25 cell lines and 24 h for fibroblasts.
but without being cytotoxic, since IC50 was greater than 50%
The data were analyzed using Prisma®GraphPad software
(Fig. 4C).
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. 3.4. Piper cubeba total extract and isolated lignans decrease cell
migration

3. Results
In the Hep-2 cell line, treatments with lignans (cubebin, dihy-
drocubebin, ethylcubebin, hinokinin), total extract and podo-
3.1. Piper cubeba total extract and isolated lignans
phyllotoxin decreased the migration of these cells in relation to the
control, statistically. In fact, the podophyllotoxin, used as a positive
After extraction with ethanol, 500 g of the powered P. cubeba
control, showed the greatest decrease in cell migration. Only the
fruit furnished 78 g of crude ethanolic extract (15% yield).
methylcubebin does not present significant statistical difference
Sequential silica gel columns chromatography produced the lignans
(Fig. 5A).
ethylcubebin (0.030 g), methylcubebin (0.022 g), hinokinin
For the SCC-25 cell line, a significant decrease in cell migration
(0.080 g), cubebin (0.072 g) and dihydrocubebin (0.060 g). Results
after treatment with the lignans dihydrocubebin, ethylcubebin,
of the NMR analysis (supplementary data) confirmed the structures
methylcubebin and podophyllotoxin was observed in relation to
of these compounds (Fig. 1). The NMR data obtained for these
the control, based on the statistical test. We can also observe that
compounds are in agreement with literature data.
cubebin, hinokinin and total extract reduced the cellular migration
rate, but did not present significant difference in relation to the
3.2. Piper cubeba total extract and isolated lignans decrease control (Fig. 5B).
proliferation without modifying cellular morphology In the fibroblasts, it was observed that only the lignan ethyl-
cubebin exerted a smaller migratory effect when compared to the
The Hep-2, SCC-25 and fibroblasts cells did not change their control statistically. However, cubebin, dihydrocubebin, methyl-
morphology characters, after the treatment with all the lignans, cubebin and podophyllotoxin showed a higher number of migrated
total extract and podophyllotoxin, when compared to the control cells than the control, probably these lignans have a low action on
group (Fig. 2). But, in the proliferation assay, all the compounds cell migration in the normal cells. The hinokinin and total extract

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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. 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- their nuclei than the control cells (Fig. 5D). In relation to the SCC-
ference in relation to the control (Fig. 5C). 25 cell line, all lignans, total extract and podophyllotoxin caused
significant damage to the nuclei of these treated cells compared to
3.5. Damage to the genetic material of the cells can be caused by the control (Fig. 5E).
the action of P. cubeba total extract and isolated lignans In the fibroblasts, only cubebin and methylcubebin generated
more lesions to the DNA, statistically, than in the control cells. The
For the Hep-2 cell line, we can observe that cubebin and damage index of the cells treated with others compounds were
podophyllotoxin presented higher significant indices of damage in 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 treated with the different lignans, showed increased expression,
expression generally, of the PTGS2, PTGER3, MMP2 and MMP9 genes and
decreased PTGER4 gene compared to the untreated control group.
The Hep-2 cell line (Fig. 6A), when treated with the different
lignans, total extract and podophyllotoxin showed increased 3.7. Lignans from P. cubeba alter the expression of PTGS2 and
expression of the PTGER3, PTGER4 and MMP9 genes and decreased MMP2 proteins in head and neck cancer cells
PTGS2 and MMP2 genes compared to the untreated control group.
The SCC-25 cell line (Fig. 6B), when treated with the different The expression of PTGS2 and MMP2 proteins were examined
compounds, showed increased expression of PTGS2 and MMP9, using the Western blot technique to evaluate the effects of cubebin
genes and decreased PTGER3, PTGER4 and MMP2 genes compared to and methylcubebin lignans, as well as podophyllotoxin, as these
the untreated control group. Normal oral fibroblasts (Fig. 6C), when compounds had greater effects on head and neck cancer cells in
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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. 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, cubebin and methylcubebin, and for the treatment with podo-
treatment with cubebin and podophyllotoxin can significantly phyllotoxin we found that there was a decrease in the protein
decrease the levels of PTGS2 protein compared to the control. expression of MMP2, but without being statistically significant in
Regarding the SCC-25 cell line, treatment with lignan methyl- relation to control.
cubebin significantly reduced the expression of PTGS2 protein
when compared to the untreated control. These data suggest that 4. Discussion
lignans modify the expression of PTGS2 protein levels in head and
neck tumorigenic cells. Regarding the MMP2 protein, cubebin lig- In the present work, we observed that the morphology of Hep-2,
nan also significantly decreased the expression of this protein in the SCC-25 and fibroblasts cells did not change after the treatments
SCC-25 lineage. As for the Hep-2 cell line, we observed a higher with total extract and isolated lignans from P. cubeba fruits. Despite
expression of MMP2 protein from treatment with the lignans this, we observed a decrease in cell proliferation after the

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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. 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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treatments in these cells, evidencing the involvement of these with cubebin significantly decreases the level of PTGS2 protein
lignans in antiproliferation mechanisms, as well as Cunha and expression in Hep-2 laryngeal cancer cells. In previous studies it was
colleagues9 also verified the antiproliferative effect of hinokinin seen that cubebin can act by inhibiting cyclooxygenase-2 (PTGS2) in
combined with doxorubicin in breast cancer cell lines (MCF-7 and rat paw edema using prostaglandin as a mediator.24 This decrease in
SKBR-3) by modulation of the cyclin-dependent kinase inhibitor 1. PTGS2 protein expression was also observed when treatment with
The cubebin also inhibited proliferation in human prostate cancer methylcubebin was performed in SCC-25 tongue cells. Thus, cubebin
cells (LNCaP) by reducing DNA synthesis and inducing apoptosis, and methylcubebin lignans decrease PTGS2 protein expression in
thus this lignan has an antitumor action as a biological activity.18 head and neck cancer cells in the present study.
In the cytotoxicity experiment, by the MTS assay, it was found The PTGER3 gene presented low expression after the lignans
that these lignans show little cytotoxicity for the Hep-2, SCC-25 and treatments and total extract in the SCC-25 cells, differently in Hep-2
fibroblast cells. That is, the lignans structural differences extracted and fibroblast cells, in which this gene was up regulated. These
from P. cubeba do not lead to significant differences on the cyto- differences in expression of the PTGER3 gene for the cells studied
toxicity observed on the three types of cells evaluated. That it is may be partially related to the isoforms of this gene, since these
good, because in the tumoral microenvironment, there are normal mainly signal the Gi (inhibitory) proteins, but also, partially, by the
and tumor cells in the same place, then if the drug is not cytotoxic is Ca2þ phospholipase cascade or the Gs (stimulatory) proteins.25
good to the patient and probably the cell's dead are not occur As expected, the PTGER4 gene showed low expression in SCC-25
because the compound is toxic, but probably this occur for another and fibroblast cells after the treatments. In the Hep-2 cell line, the
way. Of course, if these compounds were cytotoxic to neoplastic expression of this gene was up regulated. In this way, the inacti-
cells and only death of these cells would be better, but lignans were vation or negative expression of the PTGER4 receptor promotes
not cytotoxic to any cells, except fibroblasts when treated with tumorigenic reduction effects, and may be caused by blocking the
cubebin at concentration of 10 mg/mL, which was cytotoxic due to survival of PGE2-dependent tumor cells, or due to a reduction in
its IC50 being below 50%. According to Rajalekshmi et al. (2016), the blood supply necessary for oxygenation, proliferation and sur-
cubebin and its synthetic derivatives, also showed cytotoxicity ef- vival of tumor cells.26 Studies on the roles of PTGS2 receptors and
fect against different human cancer cell lines, by MTT assay.19 PTGERs in head and neck carcinomas have revealed a significant
With respect to the cell migration assay, it was found that increase in PTGS2 expression as well as all PTGER receptors,27 but
lignans promoted an anti-migratory effect in the Hep-2 and SCC-25 when the treatments (dihydrocubebin, total extract, hinokinin,
and fibroblasts cells, in which the same was not observed by da methylcubebin and podophyllotoxin) reduce the PTGER4 expres-
Silva et al.20 where the compounds derived from cubebin lignan did sion means that the treatment is acting. Then, we can suppose that
not exhibit activity in the cell migration test, investigated using these lignans could be effective in the treatment of the tongue
different animal models such as rats and mice. squamous cell carcinoma, like in the SCC-25 cells, but not so
Interestingly, ethylcubebin had an anti-migratory effect even on effective in larynx squamous cell carcinoma, like Hep-2 cells.
fibroblasts, which can be explained as it is the most hydrophobic Regarding matrix metalloproteinases, we found a lower expres-
among the lignans evaluated, that is, originally it may be easier to sion of MMP2 gene in Hep-2 and SCC-25 cell lines, and a greater
penetrate into healthy cells and with greater regularity in the shape expression in fibroblasts. The expression level of MMP2 protein was
than the other cell lines studied. also reduced in SCC-25 treated with cubebin, methylcubebin and
In our genotoxicity study, some lignans caused damage to Hep- podophyllotoxin, seen in the Western blot assay. This diminished
2, SCC-25 and fibroblast nuclei. Other authors analyzing mouse MMP2 gene and protein expression in the SCC-25 tumorigenic cell
cells, observed that the cubebin also produced genotoxic effects, line may be related to our results of the antimigratory effect of the
investigated by micronuclei (MN) assays on bone marrow cells and lignans and total extract studied, because it is known that different
comet assay on peripheral blood lymphocytes.21 In the study by groups of proteolytic enzymes are involved in the matrix breakdown
Resende et al.4 however, it was observed that lignan hinokinin had and among them, the most important group in tumor invasion and
no genotoxic effect seen by the comet assay on Chinese hamster metastasis are the genes MMPs.28 Even as in our research, the lignans
lung fibroblast cell (V79), an effect also seen on Hep-2 and normal and total extract of P. cubeba down regulated MMP2 and MMP9 gene
fibroblasts cells in our study. expression and had an antimigratory effect.
In order to complete our investigation of the effect of P. cubeba For metalloproteinase MMP9, an increase in its expression was
extract and lignans on the inflammatory response in Hep-2, SCC-25 observed in all cells analyzed. In contrast to the study by Mali
and fibroblast cells, we verified the expression of five genes (PTGS2, et al.29 in which they investigated the action of enterolactone, they
PTGER3, PTGER4, MMP2 and MMP9) related to inflammation pro- found that it significantly reduced the expression of the MMP2 and
cesses. We also analyzed PTGS2 and MMP2 proteins by Western MMP9 genes in MCF-7 and MDA MB 231 breast cancer cells.
blot assay for cubebin, methylcubebin and podophyllotoxin lignans, Probably not all the metalloproteinases undergo the effects exerted
these compounds being the ones that showed anti-tumorigenic by the lignans in the cells studied by our group.
effects through antiproliferative, antimigratory and genotoxic ac- The lignans extracted from P. cubeba have structural differences
tion on head and neck cancer cells. Hep-2 cell line showed the only in relation to the 4-membered ring, where in cubebin this ring
reduction of PTGS2 gene expression after treatment with P. cubeba is a lactol ring, in hynokinin is a lactone, in methylcubebin and
extract and lignans, but in the SCC-25 and fibroblast cells, the ethylcubebin is a ketal, while in dihydrocubebin there is a linked
lignans act to increase the expression of this gene. PTGS2 is an CH2OH group to the carbons where the ring binds to the other
inducible expression enzyme responsible for the production of lignans (Fig. 1). These structural differences alter the degree of
prostaglandins at sites of inflammation, playing a role in the initi- solubility, lipophilicity, greater or lesser rigidity of the molecule and
ation and progression of various human diseases.22 In addition, the affect the way these lignans interact in the cells, leading to differ-
PTGS2 gene is often overexpressed in a variety of human neoplasms, ences or similarities in their biological properties as observed. This
including laryngeal carcinoma.23 4-membered ring, or the absence of it in dihydrocubebin, makes
Thus, our findings with the Hep-2 laryngeal neoplastic lineage the molecules assume different conformations that can have
show that treatment with compounds extracted from P. cubeba can different models of interaction with the proteins that act in these
regulate PTGS2 expression, making it less expressed. In this sense metabolic pathways, inhibiting or enhancing the expression of the
and as expected, our Western blot results revealed that treatment studied genes.
101
 ~o et al.
J.P. Gusson-Zanetoni, J.S.G.M. da Silva, T.B. Pica Journal of Pharmacological Sciences 148 (2022) 93e102

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From these data, we can conclude that the different lignans do doi.org/10.1590/1983-084X/14_085.
not alter the cellular morphology, but they diminish the prolifera- 8. Niwa AM, de Paula NA, Vesenick DC, et al. Evaluation of lignan (e)-cubebin
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that the lignans cubebin and methylcubebin had a greater effect on contributes to the antiproliferative effects of doxorubicin in breast cancer cells.
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Toniol Cardin: Methodology. Janesly Prates: Methodology. Stefanie
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Oliveira Sousa: Methodology. Tiago Henrique: Methodology and Available at:http://repositorio.unicamp.br/jspui/bitstream/REPOSIP/312537/1/
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This work was supported by the Foundation for Research Sup- time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods.
~o Paulo - FAPESP (2017/02100e3 to FCR-L);
port of the State of Sa 2001;25:402e408. https://doi.org/10.1006/meth.2001.1262.
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Declaration of competing interest
Bioorg Med Chem Lett. 2016;26:1767e1771. https://doi.org/10.1016/
j.bmcl.2016.02.041.
The authors declared that they have no conflict of interest. 20. Da Silva R, de Souza GH, da Silva AA, et al. Synthesis and biological activity
evaluation of lignan lactones derived from (-)-cubebin. Bioorg Med Chem Lett.
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Acknowledgments 21. Maistro EL, Natel AV, Souza GH, Perazzo FF. Genotoxic effects of cubebin in
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22. Shi C, Guan Y, Zeng L, et al. High COX-2 expression contributes to a poor
pinas/UNICAMP, Piracicaba, SP, for providing primary cells derived prognosis through the inhibition of chemotherapy-induced senescence in
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Appendix A. Supplementary data oxygenase (COX)-2 inhibits proliferation, reduces invasion and enhances
chemosensitivity in laryngeal carcinoma cells. Mol Cell Biochem. 2008;317:
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