[Short title + Author Name - P&H title] 32 (2024) 101892

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Saudi Pharmaceutical Journal

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Pentagamavunone-1 inhibits aggressive breast cancer cell proliferation

through mitotic catastrophe and ROS-mediated activities: in vitro and in
vivo studies
Dhania Novitasari a, Ikuko Nakamae b, Riris Istighfari Jenie a, c, Noriko Yoneda-Kato b, Jun-
ya Kato b, *, 1, Edy Meiyanto a, c, *, 1
a
Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
b
Laboratory of Tumor Cell Biology, Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Nara 630-
0192, Japan
c
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia

A R T I C L E I N F O A B S T R A C T

Keywords: Pentagamavunone-1 (PGV-1), an analog of curcumin, has been studied for its cytotoxic effects in 4T1, MCF7,
Curcumin analog MCF7/HER2, and T47D breast cancer cells. Its antiproliferative effect is partly mediated through G2/M arrest;
Mitotic arrest however, its molecular mechanism during cell cycle progression remains unknown. In this study, we aimed to
ROS generation
determine whether PGV-1 has any anticancer effects on highly aggressive breast cancer cells, with a focus on cell
Breast cancer
cycle regulatory activity, reactive oxygen species (ROS) generation, and their mediated effects on cancer cells.
MDA-MB-231 (triple-negative) and HCC1954 (overexpressed HER2) immortalized human breast cancer cells
were used in the study. PGV-1 exhibited cytotoxic activity with an irreversible antiproliferative impact on treated
cells and had good selectivity when tested in fibroblast cells. Oral PGV-1 administration suppressed tumor
growth in a cell-derived xenograft mouse model. PGV-1 induced the phosphorylation of Aurora A kinase and
PLK1 in MDA-MB-231 cells, while PLK1 and cyclin B1 phosphorylation were enhanced in the PGV-1-treated
HCC1954 cells during prometaphase arrest. Intracellular ROS production was substantially higher upon PGV-1
treatment following mitotic arrest, and this activity caused impairment of mitochondrial respiration, induced
senescence, and subsequently triggered early-to-late apoptosis. Collectively, these results suggest that the mo­
lecular mechanism of PGV-1 involves the regulation of mitotic kinases to cause cell cycle arrest and the
enhancement of ROS production to impair mitochondrial activity and induce cellular senescence. The thera­
peutic activities demonstrated by PGV-1 in this study show its potential as an appealing candidate for chemo­
therapy in breast cancer treatment.

1. Introduction 2015), cell cycle regulation (Lashen et al., 2021), and other clinical and
histopathological aspects, which makes the treatment options compli­
Breast cancer, the most common cancer in women worldwide, is cated (Guo et al., 2023). Along with the available types of anticancer
commonly classified as either luminal, HER2-positive, or triple-negative chemotherapy used in clinical settings, antimitotics (taxanes and/or
breast cancer (TNBC) (Makanjuola et al., 2014). The triple-negative vinca alkaloids) and anthracycline doxorubicin are two of the most
subtype constitutes approximately 15–20% of the total reported breast frequently prescribed first-line medications for early breast cancer (Shen
cancer cases (Damaskos et al., 2019), while cases characterized by the and Liu, 2023). Despite their significant efficacy, the evident adverse
overexpression of the HER2 protein are observed in 20–30% of cases effects that occur following treatment with these medications make the
(Wahler and Suh, 2015). Both subtypes are notable for their aggres­ chemoresistance of breast tumors toward these drugs a major hurdle
siveness due to high heterogeneity in metabolism (Willmann et al., that can lead to failed treatment (Lovitt et al., 2018; Wanifuchi-Endo

Peer review under responsibility of King Saud University

* Corresponding authors at: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia (E. Meiyanto).
E-mail addresses: [email protected] (J.-y. Kato), [email protected] (E. Meiyanto).
1
Jun-ya Kato and Edy Meiyanto have equally contributed on the manuscript as corresponding author.

https://doi.org/10.1016/j.jsps.2023.101892
Received 12 May 2023; Accepted 1 December 2023
Available online 2 December 2023
1319-0164/© 2023 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
D. Novitasari et al. Saudi Pharmaceutical Journal 32 (2024) 101892

et al., 2022). As a result, substantial research has been undertaken to Chemical, Japan). Adult dermal fibroblasts (NHDF) were purchased
identify a rational strategy with several targets for eliminating aggres­ from Promo Cell, Germany (Cat #C-12302) and were cultured in
sive breast tumors. fibroblast growth medium (Cat #C-23020) according to the manufac­
Pentagamavunone-1 (PGV-1) (2,5-bis-(4-hydroxy-3,5-dime­ turer’s instructions. All cells were stored in a 37 ◦ C, 5% CO2, humidified
thylbenzylidene)-cyclopentanone) is an analog of curcumin that exhibits incubator.
improved efficacy in eliminating different types of cancer cells
compared to curcumin (Meiyanto et al., 2021, 2018). The effects of PGV-
1 treatment on cell growth are partly caused by G2/M arrest, which 2.2. Growth curve analysis
results in senescence and apoptosis (Meiyanto et al., 2019; Novitasari
et al., 2023b). PGV-1 inhibits cell migration by decreasing MMP–9 A total of 2 × 104 cells were plated into 48-well plates and subse­
expression, although HER2 expression remains unchanged (Meiyanto quently exposed to various concentrations of PGV-1. Following a 96 h
et al., 2021; Novitasari et al., 2021a, 2022). PGV-1 was also demon­ incubation period, cells were collected using trypsin–EDTA, and then
strated to inhibit tumor growth in vivo in a preclinical setting using stained with 0.4% trypan blue (Fujifilm Wako Chemical, Japan, Cat
xenograft models (Kamitani et al., 2022). #204-21102) so the viable cells could be counted. The percentage of
Recent studies have indicated that PGV-1 causes prometaphase ar­ living cells and the growth inhibitory effect at the 50% (GI50) value were
rest, as evidenced by the disruption of the nuclear envelope detected via determined for each cell line.
DNA staining (Lestari et al., 2019; Moordiani et al., 2023). During To determine the selectivity index (SI) score, the GI50 value in NHDF
mitosis, the activation of mitotic proteins, such as polo-like kinase 1 cells was divided by the GI50 value of the cancer cells (Indrayanto et al.,
(PLK1), Aurora A, and cyclin B1, governs the progression until cytoki­ 2021; López-Lázaro, 2015). According to Weerapreeyakul et al. (2012)
nesis. These protein kinases are at their peak from mitotic entry to and Bézivin et al. (2003), an SI value larger than 3 categorizes a sub­
metaphase (Choi et al., 2011; Fu et al., 2007; Schmucker and Sumara, stance as a potential anticancer drug.
2014). The most recent work using bioinformatic analysis revealed that The cells were exposed to PGV-1 for 72 h before being drug-washed
PGV-1 may target mitotic proteins (aurora A, cyclin-dependent kinase 1, and replaced with new medium (without PGV-1). For the subsequent 48
WEE1, etc.), which probably contributes to its ability to induce mitotic h, the cells were kept in incubators. Trypan blue exclusion was per­
arrest (Meiyanto et al., 2022). While it is well documented that PGV-1 formed daily to determine the number of living and dead cells (Lestari
causes prometaphase arrest, the specific molecular mechanism behind et al., 2019).
this phenomenon is currently unknown. In addition, PGV-1 interacts
with NQO2 and GLO1 metabolizing enzymes, resulting in an imbalanced
2.3. Cell cycle analysis
reactive oxygen species (ROS) level that may contribute to cancer cell
death (Lestari et al., 2019; Meiyanto et al., 2019). The concern about
The cells were incubated with PGV-1 for durations of 24, 48, and 72
oxidative stress levels in tumor cells is understandable because tumor
h. Propidium iodide (PI) solution (Sigma-Aldrich, USA) was used to stain
cells have higher ROS levels to maintain their metabolism and support
the cells after they were harvested at the end of the treatment. Cells were
uncontrollable proliferation, while the ROS itself can trigger anti­
filtered and collected in a tube, and then analyzed using a FACSCalibur
tumorigenic signaling and initiate oxidative stress-induced tumor cell
(BD Biosciences) flow cytometer to determine the cell cycle phase dis­
death (Arfin et al., 2021). Because the majority of ROS production oc­
tribution (Larasati et al., 2018).
curs in mitochondria as a result of the oxidative phosphorylation
(OXPHOS) process (Dong and Neuzil, 2019; Starkov, 2008), we expect
that PGV-1 treatment will also affect mitochondrial function in cancer 2.4. Double staining with annexin v and PI
cells.
This work intends to assess the cellular activities of PGV-1 in breast After 72 h treatment with PGV-1, cells were harvested by trypsini­
cancer cells in vitro and in vivo in order to gain a better understanding of zation and collected for the annexin V/PI binding assay. Cells were
its molecular mechanism in cell cycle progression. Its effects were resuspended in 100 µL of binding buffer, double-labeled with FITC-
observed using TNBC and HER2-positive cell lines, which represent annexin V and PI (BD Pharmingen, USA, Cat #556547), and incubated
aggressive breast cancer cells. This work highlights the effect of PGV-1 for 10 min. Immediately after the addition of 200 μL of 1 × binding
on the progression of the cell cycle, which involves the activities of buffer, cells were analyzed using a FACSCalibur flow cytometer (BD
key mitotic kinases (Aurora A, PLK1, and cyclin B1), leading to prom­ Biosciences) (Novitasari et al., 2021b).
etaphase arrest. Furthermore, PGV-1 treatment impairs mitochondrial
respiration while enhancing ROS formation due to mitotic arrest. Oral
PGV-1 treatment can prevent tumor growth in the cell-derived xenograft 2.5. Mitotic index determination
model. The present study highlights PGV-1 as a promising chemother­
apeutic option for the treatment of breast cancer. Following 24 h of treatment with 2 µM PGV-1, the cells were
collected and incubated for 6 min in 5.6% KCl. Upon centrifugation, the
2. Materials and methods cells were preserved in a 1:3 v/v combination of acetic acid and meth­
anol and carefully dropped on sterile microscope slides. Hoechst 33324
2.1. Compounds and cell lines (Cell Signaling Technology, USA, Cat #4082) was added to the slides,
and the slides were incubated for 1 h prior to observation under a
PGV-1 was obtained and analyzed at the Cancer Chemoprevention confocal microscope (Leica Zeiss LSM710) (Lestari et al., 2019). The
Research Center, Universitas Gadjah Mada (Indonesia). Curcumin mitotic index was determined by dividing the number of cells that had
(>65%, Sigma-Aldrich, USA, Cat #C1386) and doxorubicin hydro­ undergone mitosis by the total number of cells.
chloride (>90%, Fujifilm Wako Chemical, Japan, Cat #040-21521), as Separately, the cells on the slide were treated for 5 min with May-
reference chemotherapy for breast cancer cells, were purchased Grünwald (Sigma-Aldrich, Germany, Cat #63590). The slide was rinsed
commercially. briefly in phosphate buffer before being immersed in Giemsa’s azur
The MDA-MB-231 and HCC1954 cell lines were obtained from the solution (Sigma-Aldrich, Germany, #1.09204.0103) for 20 min. The
American Type Culture Collection (ATCC, USA) and cultured in DMEM slide was then cleaned with phosphate buffer for 1.5 min. Following a
and RPMI 1640, respectively. All media contained 10% fetal bovine quick rinse in deionized water, the slides were examined under phase-
serum (Hyclone, USA) and 1% penicillin/streptomycin (Fujifilm Wako contrast microscopy (Lestari et al., 2019).

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D. Novitasari et al. Saudi Pharmaceutical Journal 32 (2024) 101892

2.6. Immunoblotting 2.10. Xenograft assay

Whole-cell lysates were prepared from the cells treated with 2 µM Female nude mice (nu/nu) were used for this experiment. Nude mice
PGV-1 (24 h) by collecting them in a lysis buffer for protein extraction. are characterized as hairless mice with a lack of thymus; therefore, they
The lysates were denatured before being loaded onto 10% SDS-PAGE have defects in immune function due to T cell deficiency (McDermott-
gel, then blotted onto a PVDF membrane. The blots were blocked with Lancaster et al., 1987). Due to their high vulnerability to bacterial and
5% BSA prior to incubation with the target antibodies. All the phos­ viral pathogens, the mice were kept in specific-pathogen-free animal
phorylated antibodies, including phospho-cyclin B1 (Cat #4133), house. The animal house had an equal light–dark cycle, a constant
phospho-PLK1 (Cat #9062), and phospho-Aurora A kinase (Cat #3079), temperature ranging from 23 ◦ C to 24 ◦ C, and 50–70% humidity. Mice
were obtained from Cell Signaling Technology (USA); as well as total had free access to autoclaved water and sterile food. The NAIST Insti­
protein of Aurora A kinase (Cat #12100) and β-actin (Cat #3700). PLK-1 tutional Animal Care and Use Committee approved the protocol for the
antibody was purchased from Abcam (USA, Cat #ab17056) and cyclin relevant aspects of the animal-related experimental tests (reference
B1 was obtained from Santa Cruz (Cat #sc-752). The antibodies were number: 1805).
dissolved in 5% BSA in PBS (with 0.05% sodium azide) (1:1000 v/v), Subcutaneous injections of 1 × 107 cells (in 0.1 mL of culture me­
and phosphorylated antibodies were dissolved in Can Get Signal solu­ dium) were made into the flanks of the mice on day 0. Simultaneously,
tion (Toyobo, Japan, Cat #NKB-101) (1:500 v/v). After probing with the the mice were treated orally with 25 mg/kg BW PGV–1, as reported in
primary antibody, the membrane was incubated with secondary anti­ the study by Kamitani et al. (2022). PGV-1 was administered orally
bodies (Cytiva, UK) for antimouse (Cat #NA931V) or antiprotein A (Cat every 2 days, and the tumor size was recorded during that time. The
#NA9120V). Detection was performed using enhanced chem­ tumor was excised at the completion of the experiment and weighed
iluminescence (ECL) (Cytiva, UK, Cat #RPN2106), and the protein using an analytical balance.
bands were exposed using x-ray film (Fujifilm, Japan, Cat
#4741026617). The intensity of each protein band was quantified using 2.11. Statistical analysis
ImageJ software (NIH, USA). The expression of each target protein was
analyzed by comparing it with the expression of β-actin (with normali­ Statistical analysis was conducted using the unpaired T-test. A p
zation expressed as the fold change relative to the untreated group), value of < 0.05 was considered statistically significant, and the graphs
then the ratio of phosphorylated protein to total protein was calculated. were created in GraphPad (version 9.0). Results were reported as the
mean ± standard deviation (SD). All p-values were inserted in each
figure.
2.7. Senescence assay
3. Results
Following 24 h treatment with PGV-1, the cells were fixed in 4%
formaldehyde for 10 min and washed with 1 × PBS. Then, 0.2% X-gal 3.1. PGV-1 exhibits cytotoxic and antiproliferative effects on breast
solution was added to the cells, and the cells were incubated before cancer cells
being observed under phase-contrast microscopy (Debacq-Chainiaux
et al., 2009). The antiproliferative properties of PGV-1 were assessed in MDA-MB-
231 (triple-negative) (Fig. 1A), HCC1954 (HER2-positive) (Fig. 1B), and
NHDF (fibroblast) cells (Fig. 1C). Following a 96 h incubation with PGV-
2.8. Intracellular ROS level measurement 1 (dosage range of 0.001–10 µM), PGV-1 exhibited a substantial cyto­
toxic effect, with a GI50 score of 0.9 ± 0.46 µM for MDA-MB-231 cells,
Prior to the treatment, the cell suspension was added to 270 µL of 0.4 ± 0.12 µM for HCC1954 cells, and 3.5 ± 1.08 µM for NHDF cells
supplemented buffer (containing 10% FBS in 1 × PBS) and stained with (Fig. 1D). The similar GI50 values in the MDA–MB–231 and HCC1954
30 µL of 2′,7′-dichlorofluorescin diacetate (DCFDA) solution (final con­ cells indicated that breast cancer cells are sensitive to PGV-1.
centration: 20 µM) (Sigma-Aldrich, USA, Cat #D6883). The cells were We also tested the same cell lines with curcumin and the chemo­
stored in an incubator for 30 min, then PGV-1 was added to the therapy drug doxorubicin (a common option for breast cancer therapy).
microtube. The microtube was placed back in the incubator, which was While doxorubicin was found to be the strongest agent to eliminate these
set for three interval times (4, 8, and 24 h). The ROS intensity was cancer cells, the antiproliferative profile of PGV-1 demonstrated simi­
determined using flow cytometry, then the mean fluorescence was larity to that of curcumin in HCC1954 cells. However, in MDA-MB-231
normalized against the untreated group (Sarmoko et al., 2023). cells, PGV-1 was revealed to be three times more potent than curcumin.
PGV-1 also demonstrated low antiproliferative activity when tested in
normal fibroblast cells, demonstrating its selectivity for cancer cells,
2.9. Mitochondria respiration analysis with a SI value greater than 3 (Fig. 1D). Based on these results, we
attempted to observe the cellular and molecular activities of PGV-1 in
Cells were treated with PGV-1 for 24 h in a 35-mm culture dish. MDA-MB-231 and HCC1954 cells.
Following trypsinization, MDA–MB-231 (2.25 × 105 cells) and PGV-1 suppressed MDA-MB-231 cell growth (p < 0.0001) and
HCC1954 (1.5 × 105 cells) cells were dissolved in assay medium con­ induced cell death (p < 0.05) after 96 h of treatment (Fig. 2A). PGV-1
taining 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate and also had an antiproliferative effect in HCC1954 cells, as evidenced by
equally distributed in a Cell-Tak (Corning, USA, Cat # 354240) pre­ a significant reduction in viable cells (p < 0.001), followed by an
coated miniplate. After spinning down the 8–well plate for cell adhesion, increasing percentage of dead cells (p < 0.05) up to the 4th day (Fig. 2B).
it was incubated for 30 min. Mito test kit reagents (Agilent, Germany, Owing to the high chance of treatment failure in breast cancer therapy
Cat #103010–100) containing FCCP (1 µM), oligomycin (1.5 µM), and due to relapse, PGV-1 was cultured in cell medium for 72 h before being
rotenone/antimycin (0.5 µM) were injected into the cartridge, then the replaced without the compound. A lack of expansion in viable MDA-MB-
oxygen consumption rate (OCR) was recorded using an Agilent XF HS 231 cells with a steadily rising percentage of dead cells (p < 0.01)
mini analyzer. Finally, the cells were measured for total protein to suggested that PGV-1 irreversibly inhibited cell growth (Fig. 2C). Even
normalize the OCR data and later analyzed using the Seahorse Analytics after removal of PGV-1 from the medium, cell growth suppression was
platform (https://seahorseanalytics.agilent.com/#) (Novitasari et al., still evident, and HCC1954 cells gradually lost their viability (p < 0.01)
2023a; Reda et al., 2019). (Fig. 2D). Therefore, PGV-1 demonstrates an irreversible

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Fig. 1. PGV-1 inhibits breast cancer cell growth. (A) MDA-MB-231 and (B) HCC1954 cells growth curves upon PGV-1 treatment, along with curcumin and
doxorubicin as reference compounds (C) The growth from NHDF cells that were treated with PGV-1 (D) The GI50 and selectivity index (SI) values of PGV-1. Results
are expressed as mean ± SD of triplicate samples.

antiproliferative effect on breast cancer cells. 231 cells, suggesting that PGV-1 may trigger apoptosis. We also
noticed an increasing percentage in the double-positive (Anx+/PI+)
population (p < 0.0001) after PGV-1 exposure in HCC1954 cells. How­
3.2. PGV-1 triggers mitotic arrest in breast cancer cells ever, Anx− /PI+ cells were also detected in the HCC1954 cells (Fig. 4D),
indicating damage to the cell membrane, enabling PI to bind with DNA.
To achieve a better understanding of how PGV-1 hinders cell growth, These results support the notion that PGV-1 has the potential to induce
we exposed cells to 2 µM PGV-1 for 24, 48, and 72 h and analyzed their apoptosis in breast cancer cells.
cell cycle profiles. PGV-1 halted MDA-MB-231 cells at G2/M within the Because PGV-1 induced G2/M arrest, a follow-up experiment was
first 24 h (51.8% ± 9.4% versus 19.6% ± 1.2% of untreated cells, p < conducted using chromosomal staining to determine whether PGV-1
0.01) (Fig. 3A and 3B). Exposure to PGV-1 prompted the cells to arrest in halted the cell cycle at the Gap2 (G2) phase or the mitosis phase. The
G2/M, then gradually shift into hyperploid stage arrest (7.4% ± 3.5% at observations from the Giemsa staining demonstrated that PGV-1 caused
24 h versus 14.3% ± 15.0% at 72 h), indicating the failure of cell divi­ aberrant chromosomal condensation and disappearance of the nuclear
sion. The subG1 cell population was also found to be increased in the envelope membrane, indicating that many cells were arrested at mitosis
PGV–1-treated group at the end of the observation (11.7% ± 6.0% (Fig. 5A). The quantification analysis revealed that PGV-1 dramatically
versus 4.9% ± 4.1% in the untreated group at 72 h). In HCC1954 cells, (p < 0.01) increased the percentage of mitotic cells from 3.7% ± 2.7% to
PGV-1 caused G2/M arrest (37.8% ± 7.8% versus 23.9 ± 3.6% in the 15.1% ± 2.1%. Similarly, PGV-1 increased (p < 0.05) the number of
untreated group, p < 0.05) after the first 24 h of treatment. However, the mitotic HCC1954 cells.
effect of PGV-1 was diminished during longer exposure times (18.4% ± The chromosome was also stained with Hoechst and then viewed
5.0% at 48 h and 13.7% ± 4.4% at 72 h) (Fig. 3C and 3D). In contrast to under confocal microscopy to establish the precise mitotic stage after
MDA-MB-231 cells, hyperploid HCC1954 cells were absent following PGV-1 treatment. PGV-1 caused prometaphase arrest in the mitotic
PGV-1 treatment. This phenomenon was followed by cell accumulation phase, as indicated by chromosomal condensation and segregation upon
at subG1 in PGV-1-treated HCC1954 cells, starting at 15.1% ± 12.1% at nuclear envelope breakdown, meaning the cells could not properly
24 h and reaching 41.8% ± 24.2% after 72 h, which was 2.7 times move to the equatorial plane (Fig. 5B). The results of the mitotic index
higher than the untreated group (15.3% ± 4.3% at 72 h). These data calculation showed that PGV-1 significantly (p < 0.05) induced mitotic
imply that PGV-1 inhibits cell growth mainly during G2/M phase. cell formation (17.5 ± 3.5) compared to the untreated group (0.3 ± 0.5)
Since 72 h posttreatment showed the cell population in subG1, we in MDA-MB-231 cells. A similar finding was observed in HCC1954 cells,
determined the apoptosis incidence through double labeling with as prometaphase-arrest cells were present in the PGV-1 group, with a
annexin V (AnxV) bound to phosphatidylserine (PS) from the inner cell mitotic index of 2.8 ± 0.6, which was higher than that of the untreated
membrane and PI to stain the DNA. The flow cytogram showed that, group (0.2 ± 0.3). These results suggest that PGV-1 targets the cell cycle
after PGV-1 exposure, some MDA-MB-231 (Fig. 4A) and HCC1954 progression of breast cancer cells during prometaphase.
(Fig. 4B) cells were labeled with both AnxV and PI. The quantification
results showed that there was a drastic increase (p < 0.0001) in AnxV+/
PI+- and AnxV+/PI− -labeled cells after PGV-1 treatment in MDA-MB-

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Fig. 2. PGV-1 inhibits breast cancer cell growth in an irreversible manner. (A) The viable cells and dead MDA-MB-231 and (B) HCC1954 cells after 2 µM PGV-1
treatment for 96 h. (C) The growth curve of viable and cell death percentage of MDA-MB-231 and (D) HCC1954 cells after removal of 2 µM PGV-1 from culture
medium. Results are displayed as average ± SD (n = 3). (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

3.3. PGV-1 causes mitotic arrest through mitotic kinase activation in cancer cells.
breast cancer cells

Next, we examined the protein levels of major mitotic regulators, 3.4. PGV-1 promotes senescence in breast cancer cells
such as Aurora A kinase, PLK1, and cyclin B1, including both the total
protein and its phosphorylated form (Fig. 6A). The phosphorylation of Since PGV-1-treated cells did not perish within 24 h but were halted
Aurora A kinase was not affected in HCC1954-treated cells, in contrast in mitosis, the mechanism behind this occurrence was investigated
with the result in MDA-MB-231 cells (Fig. 6B). PLK1 phosphorylation further. Therefore, we evaluated whether PGV-1 also triggered senes­
was enhanced (p < 0.05) in both cell lines (Fig. 6C), indicating PGV-1 cence (stable proliferation arrest) in aggressive breast cancer cells. PGV-
activity during mitosis. Meanwhile, cyclin B1 activation upon PGV-1 1 treatment significantly (p < 0.05) promoted MDA-MB-231 cell
treatment was increased and notably higher (p < 0.05) in HCC1954 senescence by 30.5% ± 12.6% (Fig. 7A). A similar phenomenon was
cells than in MDA-MB-231 cells (Fig. 6D). Based on these findings, it observed in the HCC1954 cells, where PGV-1 exposure enhanced cell
appears that the mitotic arrest induced by PGV-1 is mediated by a senescence by 29.1% ± 4.0% (p < 0.001) (Fig. 7B). These findings reveal
distinct set of related regulators with respect to its activity in breast that PGV-1-induced senescence may be associated with decreased cell
viability and extended cell cycle arrest.

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Fig. 3. PGV-1 induces G2/M arrest during cell cycle progression. (A) Flow cytogram and (B) MDA-MB-231 cells distribution in each phase after treatment with 2
µM PGV-1 (C) The profile of cell cycle after 2 µM PGV-1 treatment in HCC1954 cells was followed by (D) distribution of cell accumulation in every cell cycle. Results
are showed as average ± SD (n = 3). (*p < 0.05 and **p < 0.01 compared to untreated).

3.5. PGV-1 enhances ROS production and impairs mitochondrial and Chandel, 2014). The cellular OCR was measured as a marker of
respiration mitochondrial respiration to see how PGV-1 treatment affected mito­
chondrial function (Fig. 8B). After 24 h of treatment, the maximal
Cellular senescence is commonly associated with harmful cellular respiration was suppressed (p < 0.05) in HCC1954 cells, but not in MDA-
species, such as ROS (Davalli et al., 2016). Our data showed that PGV-1 MB-231 cells (Fig. 8C). PGV-1 treatment suppressed the ATP-linked
induced cellular senescence in breast cancer cells; therefore, we respiration in MDA-MB-231 (p < 0.001) but not in HCC1954 cells
measured the ROS level to understand the correlation between these two (Fig. 8D). Also, PGV-1 treatment reduced basal respiration (p < 0.001) in
phenomena. In MDA-MB-231 cells, PGV-1 exposure caused a significant MDA-MB-231 cells; however, the level was unaltered in HCC1954 cells
increase in ROS level (p < 0.01) even after only 4 h of incubation, and (Fig. 8E). These results indicate that PGV-1 triggers mitochondrial
this elevated ROS level was maintained until 24 h. Similarly, the ROS dysfunction in breast cancer cells, possibly due to increased production
levels remained noticeably (p < 0.01) greater in the PGV-1-treated of ROS.
HCC1954 cells compared to the untreated HCC1954 cells (Fig. 8A).
These findings suggest that ROS generation is a potential cellular 3.6. PGV-1 inhibits tumor formation in xenograft models
mechanism of PGV-1 to induce senescence and mitotic arrest.
When ROS levels are elevated, mitochondria are damaged due to The antitumorigenesis effect of PGV-1 was also evaluated in vivo
changes in their respiratory chain and membrane permeability (Sullivan using a xenograft model. Athymic-nude mice bearing MDA-MB-231 or

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Fig. 4. PGV-1 triggers cell apoptosis. (A) MDA-MB-231 and (B) HCC1954 cells population after exposure of 2 µM PGV-1 for 72 h based on apoptosis quadrant after
labeling with Annexin V (AnxV) - propidium iodide (PI). Results are showed as average ± SD (n = 3). (****p < 0.0001 compared to untreated).

HCC1954 cells were established by subcutaneously injecting cells into the results reported for epithelial kidney cells (Meiyanto et al., 2021).
the flank, followed by treatment with 25 mg/kg BW PGV-1 every 2 days. These preliminary data promote further investigation of PGV-1 to
PGV-1 suppressed tumor growth (p < 0.01) (Fig. 9A), though the weight determine its underlying molecular mechanism in breast cancer cells.
of the tumor nodules were not significantly repressed in MDA-MB-231- Our results showed that PGV-1 triggers cell cycle arrest in the
xenograft mice (Fig. 9B and 9C). The study using the HCC1954 xenograft prometaphase, suggesting that, regardless of cancer cell type, PGV-1 acts
model also showed that the oral administration of 25 mg/kg BW PGV-1 similarly in inhibiting mitosis progression (Lestari et al., 2019; Moor­
effectively and significantly (p < 0.01) repressed the tumor size to 157 diani et al., 2023). Interestingly, PGV-1 promoted hyperploid formation
mm3 compared to the untreated group (992 mm3) (Fig. 9D). The tumor in MDA-MB-231 cells after prolonged incubation, indicating that mitotic
also shrank significantly (p < 0.001) after treatment with PGV-1 (95 mg failure may occur. Prolonged arrest in mitosis (notably in prometaphase
versus 290 mg in the untreated group) (Fig. 9E and 9F). These results or metaphase) can cause tetraploidization upon trigger from chemo­
confirm that PGV–1 demonstrates a tumor-suppressing effect in vivo. therapy, which prompts the cells to exit mitosis without undergoing
cytokinesis (Haschka et al., 2018). Conversely, the polyploid charac­
4. Discussion teristics were absent in HCC1954 cells following PGV-1 treatment. One
possible explanation for this finding is that a substantial number of cells
This study was designed to evaluate the anticancer activity of mon­ had already entered the subG1 phase after 24 h of PGV-1 exposure
ocarbonyl PGV-1 against highly aggressive breast cancer cells. Other (<2N), showing that HCC1954 cells are more responsive to PGV-1 than
studies employing different breast cancer cell lines, such as luminal MDA-MB-231 cells. SubG1 populations are frequently associated with
MCF-7 and T47D cells (Meiyanto et al., 2014; Wulandari et al., 2020), the occurrence of apoptosis due to reduced DNA content (Plesca et al.,
HER2-overexpressed MCF7 cells (Meiyanto et al., 2021), and triple- 2008). Since the subG1 phase is generally defined as having fragmented
negative 4 T1 cells (Meiyanto et al., 2019), have revealed the anti­ DNA content, we further evaluated this finding via Annexin V-PI double
cancer properties of PGV-1. Through this study, we were able to deter­ labeling to identify the initial stage of apoptosis. The findings demon­
mine that PGV-1 irreversibly inhibits cell proliferation in the two most strated that PGV-1 increased the proportion of early and late apoptotic
aggressive breast cancer subtypes (triple-negative MDA-MB-231 and (Annexin V-labeled) cells in MDA-MB-231 cells compared to HCC1954
HER2-amplified HCC1954), which may potentially lower the risk of cells. The presence of permeabilized HCC1954 cells (marked by AnxV− /
tumor recurrence after treatment. Therapeutic selectivity is one of the PI+) following PGV-1 treatment indicated that the treatment may induce
most crucial aspects of cancer treatment (Atkins and Gershell, 2002), cell membrane perturbation, one of the phenomena observed in cellular
and we also observed the selectivity of PGV-1 in non-cancerous cells. cytotoxicity. In this case, the membrane damage caused by PGV-1
Our results showed that PGV-1 is selective in inhibiting cancer cell caused cells to be positively stained with PI only (as seen in the popu­
proliferation, as it did not affect fibroblast cells; this is consistent with lation of AnxV− /PI+ cells). Since this technique is time-sensitive, even

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Fig. 5. PGV-1 induces prometaphase arrest during the mitosis. (A) The MDA-MB-231 and HCC1954 cells morphology upon staining with May–­
Grünwald–Giemsa (MGG), following by the calculation of %mitotic cells after 24 h incubation with 2 µM PGV-1. The appearance of mitotic cells is indicated by the
black arrow (B) The DNA-bind Hoechst 33342 labeling in treated cells, followed by quantification for mitotic index. The enlarged cells image displayed the
prometaphase-arrest cell. The results are reported as average ± SD (n = 3) (*p < 0.05 and**p < 0.01).

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Fig. 6. PGV-1 phosphorylates mitotic kinases. (A) Immunoblot presentation of PGV-1-treated lysates in MDA-MB-231 and HCC1954 cells, later followed by
quantification for the intensity of (B) phospho-Aurora A, (C) phospho-PLK1, and (D) phospho-cyclin B1 against its total protein. The histogram is displayed as average
± SD (n = 3) (ns: not significant; *p < 0.05).

live cells can be permeable to PI during a prolonged incubation period. via γ-tubulin signaling (Cowley et al., 2009; Tillery et al., 2018). Aurora
Furthermore, at an early phase of apoptosis, partial DNA content (subG1 A also facilitates γ-tubulin recruitment in prometaphase, even at low
fraction) can be positive in exposed PS residue (Darzynkiewicz et al., protein levels, due to its capability to remain active through autophos­
2001), which also explains why the proportion of the subG1 population phorylation in Thr288. However, this event can lead to centrosome
was different from the distribution in the apoptosis assay. PGV-1 is dysfunction after centriole separation breaks down (Marumoto et al.,
known to induce PUMA and BAX expression and to trigger PARP 2003). We also observed PLK1 phosphorylation in PGV-1-treated cells,
cleavage in MCF-7 cells (Da’i et al., 2017). Therefore, a different confirming its peak activity in mitosis. In centrosome maturation during
approach should be employed to assess the apoptosis incidence after prometaphase, PLK1 acts as part of the Aurora A/CEP192 axis, whereas
PGV-1 treatment. CEP192 acts as a scaffold for Aurora A and PLK1 and increases their
In another context, subG1 populations can also arise from the proximity to each other (Asteriti et al., 2015; Joukov and Nicolo, 2018;
accumulation of uneven DNA content (i.e., from hypodiploid and Park et al., 2023). Consequently, the activity of PGV-1 in prometaphase
hyperdiploid cells) due to transient mitotic arrest caused by antimitotic arrest may involve centrosome activity. Instead of being degraded, PGV-
drugs (Demidenko et al., 2008). Certain mitotic cells emerge in subG1 1 also induces the phosphorylation of cyclin B1 in breast cancer cells.
due to micronuclei or DNA fragmentation caused by mitotic catastrophe During prometaphase, cyclin B1 is located in the centromere and is
(Kawamura and Fujikawa-Yamamoto, 2009). This may also occur in concentrated in the kinetochore’s outer plate along with CDK1 (Yu and
response to PGV-1 treatment to induce mitotic catastrophe before Yao, 2008). To guarantee chromosomal adherence to the mitotic spin­
entering the apoptosis stage. dle, cells must maintain a mitotic state with persistently strong cyclin
Since this study confirms the activity of PGV-1 in inhibiting mitotic B1-CDK1 activity (Lindqvist et al., 2007). Cells that are incapable of
progression in breast cancer cells, we observed whether the mitotic ki­ completing mitosis have unusual morphological nuclear alterations,
nases were also altered by this compound. Our results showed that the which are characterized by an aberrant rise in the level of cyclin B1 and
phosphorylation of Aurora A protein was induced by PGV-1 in a delay in mitotic transition (Castedo et al., 2004). A study with anti­
MDA–MB–231 cells. During prometaphase, Aurora A participates in the mitotic nocodazole showed that binding with MAD2 (a mitotic check­
development of centrosomes and the establishment of a bipolar spindle point complex protein), which accompanied cyclin B1 upregulation,

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Fig. 7. PGV-1 promotes cellular senescence. (A) The cellular senescence observation and percentage of senescence in MDA-MB-231 (n = 9) and (B) HCC1954 cells
(n = 6) following treatment with 2 µM PGV-1 (24 h). The green-stained cells indicate senescent cells. Results are demonstrated as average ± SD (*p < 0.05 and ***p
< 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

caused prometaphase arrest in MCF7 cells (Choi et al., 2011). The cyclin overburdens mitotic cells with mitochondria that have deteriorated
B1 upregulation that was related with G2/M arrest was also observed in functionally. Mitochondria are crucial for maintaining the intrinsic
another report that used TNBC MDA-MB-468 cells treated with sesqui­ oxidative stress within tumor cells in order to sustain high cellular
terpene lactones derivates (Chimplee et al., 2022). These cascades sug­ metabolism, making them susceptible to greater oxidative stress from
gest that PGV-1 uniquely targets centromere activity during exogenous ROS-generating substances (Starkov, 2008). Moreover,
prometaphase, leading to mitotic arrest. mitochondria also fulfill a crucial function in the process of apoptosis by
Mitochondrial dysfunction and the subsequent production of facilitating the release of cytochrome c due to alterations in the
elevated ROS impose an immense burden on cancer cells that are halted permeability of the mitochondrial membrane (Chen and Lesnefsky,
in mitosis (Kumari et al., 2018; Sullivan and Chandel, 2014). Our find­ 2006). Since PGV-1 increases oxidative stress by binding to ROS-
ings revealed that in addition to PGV-1-treated cells being primarily eliminating enzymes (Lestari et al., 2019), this finding suggests a po­
arrested at M phase, this curcumin analog generates an increase in ROS tential association between PGV-1 and mitochondrial damage, which
formation. Furthermore, the effect of PGV-1 to enhance ROS formation leads to an increase in ROS production, inhibition of mitochondrial
is comparable to that of peroxide, which also escalates the ROS level respiration, and initiation of apoptosis.
(Supplementary Fig. 1A–1B) following G2/M arrest (Supplementary The significant increase in galactosidase activity following PGV-1
Fig. 1C–1D). Prior studies have shown that PGV-1 interacts with ROS- treatment is evidence that these events can cause cancer cells to expe­
metabolizing enzymes (NQO1, NQO2, and GLO1), which probably me­ rience stress-induced senescence in response to ROS exposure (Barra
diates the increased ROS level in cancer cells (Lestari et al., 2019; et al., 2022). In another context, microtubule inhibitors (vinca alkaloids,
Meiyanto et al., 2021). Wu et al. (2017) discovered that peroxide- paclitaxel, and docetaxel) that hinder mitotic arrest by targeting spindle
induced stress delays prometaphase/metaphase due to spindle assem­ dynamics may also mediate significant DNA damage and initiate
bly checkpoint activation, which is presumably also demonstrated by senescence (Bojko et al., 2019; Schwarze et al., 2005). It is important to
PGV-1 to enhance prometaphase arrest. The oxidative stress caused by remember that apoptosis typically occurs at greater concentrations of
antimitotic chemotherapeutics can have two effects: producing mitotic chemotherapeutic drugs, while the senescent cell state is initiated at
arrest (Wang et al., 2017) or overriding the mitotic spindle checkpoint, lower doses (Roninson, 2003). Although some chemotherapeutics can
resulting in premature mitotic exit with aneuploidy (D’Angiolella et al., cause senescence, the apoptotic response is prevalent in most tumors
2007). These findings corroborate the notion that PGV–1-induced (Kaufmann and Earnshaw, 2000). Through this study, we observed that
oxidative stress is linked to mitotic arrest. PGV-1 can restrain the cells during mitosis, induce senescence through a
Furthermore, PGV-1 treatment impairs mitochondrial respiration, catastrophic event, then cause cell death.
resulting in ROS accumulation and causing prometaphase arrest. This The growth of MDA-MB-231 and HCC1954 tumors in xenograft mice

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Fig. 8. PGV-1 increases the amount of ROS within the cell and impedes mitochondrial respiration. (A) The cellular ROS intensity following 2 µM PGV-1
treatment in MDA-MB-231 (n = 9) and HCC1954 cells (n = 9) (B) The graphical representation of oxygen consumption rate (OCR) is analyzed through the Sea­
horse analytics webtool. The OCR level graph is plotted alongside parameters for (C) maximal respiration, (D) ATP-linked respiration, and (E) basal respiration (n =
3). Results are illustrated as average ± SD (ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).

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Fig. 9. PGV-1 suppresses the formation of MDA-MB-231 and HCC1954 cell-derived xenograft mice. (A) Tumor volume and (B) Tumor weight from the sacrifice
of the mice bearing MDA-MB-231 tumor (C) Graph of tumor volume from HCC1954-xenograft mice (D) was plotted, along with the tumor weight (E) and the
appearance of the tumor (F) after the completion of PGV-1 treatment. Data are presented as mean ± SD (***p < 0.001; **p < 0.01; ns = not significant).

were effectively suppressed by the oral treatment of PGV-1, suggesting Ethics approval
that PGV-1 possesses antitumorigenic effects in vivo. We realize that
these subcutaneous tumors can be altered by variables such as the in­ The NAIST Institutional Animal Care and Use Committee (Japan)
jection technique, cell line, and growth rate (Brough et al., 2023; Guerin permits the use of in vivo experimental protocols for this study under the
et al., 2020), which may affect tumor size variation. Prior studies in reference number 1805.
leukemia K562-implanted mice (Lestari et al., 2019) and murine TNBC
4T1-xenograft mice (Meiyanto et al., 2021) also reported similar anti­ Funding
tumor evidence for PGV-1. Moreover, PGV-1 treatment in pancreatic
cell-derived and patient-derived xenografts have been shown to have no This work is financially supported through Master Education Leading
discernible impact on body weight, hematological profiles, or mouse to Doctoral Program for Excellent Graduate (PMDSU) (No. 2328/UN1/
behavior (Kamitani et al., 2022). No abnormalities in rat livers were DITLIT/DIT-LIT/PT/2021) and “Penelitian Dasar Kemitraan” (No.
reported based on the morphology and the expression of Ki-67 following 2119/UN1/DITLIT/Dit-Lit/PT.01.03/2023) Research Scheme from
oral administration of PGV-1 for 16 weeks (Novitasari et al., 2023b). Ministry of Education, Culture, Research, and Technology, Republic of
Furthermore, the evaluation of acute toxicity following PGV-1 treatment Indonesia.
revealed no instances of mortality or significant adverse effects (Tim
Molnas Fak. Farmasi UGM, 2001). The results of this study offer more CRediT authorship contribution statement
evidence to strengthen the effectiveness of PGV-1 therapy through oral
administration, regardless of the specific cancer type or its malignant Dhania Novitasari: Methodology, Supervision, Resources, Writing –
characteristics. Further experiments using a larger sample size are review & editing. Ikuko Nakamae: Methodology, Supervision, Re­
needed to fully comprehend how PGV-1 exerts its antitumorigenic ef­ sources, Writing – review & editing. Riris Istighfari Jenie: Methodol­
fects in vivo. ogy, Supervision, Resources, Writing – review & editing. Noriko
Yoneda-Kato: Methodology, Supervision, Resources, Writing – review
5. Conclusion & editing. Jun-ya Kato: Methodology, Supervision, Resources, Writing
– review & editing. Edy Meiyanto: Methodology, Supervision, Re­
The findings of this study suggest that PGV-1 disrupts prometaphase, sources, Writing – review & editing.
activates mitotic kinases (aurora A, PLK1, and cyclin B1), increases ROS
levels, promotes senescence, and impairs mitochondrial respiration to Declaration of Competing Interest
facilitate cell death. Because of its anticancer capabilities, PGV-1 has the
potential to become a notable antineoplastic option for malignant breast The authors declare that they have no known competing financial
cancer chemotherapy. interests or personal relationships that could have appeared to influence
the work reported in this paper.

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Appendix A. Supplementary data Guo, L., Kong, D., Liu, J., Zhan, L., Luo, L., Zheng, W., Zheng, Q., Chen, C., Sun, S., 2023.
Breast cancer heterogeneity and its implication in personalized precision therapy.
Experimental. Hematol. Oncol. 12, 3. https://doi.org/10.1186/s40164-022-00363-
Supplementary data to this article can be found online at https://doi. 1.
org/10.1016/j.jsps.2023.101892. Haschka, M., Karbon, G., Fava, L.L., Villunger, A., 2018. Perturbing mitosis for anti-
cancer therapy: is cell death the only answer? EMBO Rep 19, e45440. 10.15252/
embr.201745440.
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