Polish Journal of Medical Physics and Engineering March 2022

The Journal of Polish Society of Medical Physics Vol 28, Issue 1

ISSN 1898-0309, doi: 10.2478/pjmpe-2022-0004

Scientific Paper

Effects of Bismuth Oxide Nanoparticles, Cisplatin and Baicalein-rich

Fraction on ROS Generation in Proton Beam irradiated Human Colon
Carcinoma Cells
Noor Nabilah Talik SISIN 1,BCD, Hiroaki AKASAKA2,AB, Ryohei SASAKI2,A, Takahiro TOMINAGA3,AB,
Hayato MIURA4,B, Masashi NISHI4,B, Moshi GESO5,AE, Nor Fazila Che MAT1,AB, Khairunisak Abdul RAZAK6,AB,
Wan Nordiana RAHMAN1,ABEF,*
1School of Health Sciences, Universiti Sains Malaysia, Kelantan, Malaysia
2Division of Radiation Oncology, Kobe University Graduate School of Medicine, Kobe, Japan
3Faculty of Health Sciences, Hiroshima International University, Hiroshima, Japan
4Hyogo Ion Beam Medical Centre, Hyogo, Japan
5Medical Radiation Discipline, School Medical Sciences, RMIT University, Victoria, Australia
6School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia, Penang, Malaysia

*Corresponding author: Wan Nordiana Rahman; wandiana@usm.my

(received 12 December 2021; revised 4 March 2022; accepted 6 March 2022)

Abstract
Introduction: Proton beam radiotherapy is an advanced cancer treatment technique, which would reduce the effects of
radiation on the surrounding healthy cells. The usage of radiosensitizers in this technique might further elevate the
radiation dose towards the cancer cells.
Material and methods: The present study investigated the production of intracellular reactive oxygen species (ROS) due
to the presence of individual radiosensitizers, such as bismuth oxide nanoparticles (BiONPs), cisplatin (Cis) or baicalein-
rich fraction (BRF) from Oroxylum indicum plant, as well as their combinations, such as BiONPs-Cis (BC), BiONPs-
BRF (BB), or BiONPs-Cis-BRF (BCB), on HCT-116 colon cancer cells under proton beam radiotherapy.
Results: It was found that the ROS in the presence of Cis at 3 Gy of radiation dose was the highest, followed by BC,
BiONPs, BB, BRF, and BCB treatments. The properties of bismuth as a radical scavenger, as well as the BRF as a natural
compound, might contribute to the lower intracellular ROS induction. The ROS in the presence of Cis and BC
combination were also time-dependent and radiation dose-dependent.
Conclusions: As the prospective alternatives to the Cis, the BC combination and individual BiONPs showed the capacities
to be developed as radiosensitizers for proton beam therapy.

Keywords: ROS; bismuth oxide nanoparticles; cisplatin; proton.

Introduction tumors without harming the normal healthy tissues. 3 The

potential of proton beam therapy have led to more research and
In radiotherapy, treatment techniques using photon and electron
clinical endeavor in cancer treatment using ion beams.3-5
beams are commonly used to treat various types of malignant
Apart from that, the application of radiosensitizers in
diseases. Heavy particles such as proton beams and carbon ions
radiotherapy is also widely explored to target the effect of
have also started to be widely used in clinical settings for cancer
ionizing radiation on cancer cells while lowering side effects
treatments. In comparison to conventional photon beam, proton
onto normal cells. Many types of radiosensitizers are being
beam is of higher LET that will cause more damages to cells. 1
explored, such as chemicals or drugs, metal-based nanoparticles
Charged particles therapies have been found to cause more
(NPs), and natural compounds.6-8 Cisplatin, doxorubicin, and
damages due to double-strand breakage as well as reducing the
docetaxel are among the commercial drugs used as
cellular repairability and the cell-cycle dependency.2 Proton
radiosensitizers.9 However, the toxicities of commercial chemo-
beam radiotherapy has a broad-slow-rising entrance area before
drugs10,11 had obliged the researchers to look at non-toxic agents
the sharp-rising Bragg peak, which is useful to treat deep-seated
and radiosensitizers, which may be developed from natural

© 2022 Authors This is an open access article licensed under the Creative Commons Attribution-NonCommercial-NoDerivs License
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Authors’ contribution: A – Research concept and design, B – Collection and/or assembly of data, C – Data analysis and interpretation,
D – Writing the article, E – Critical revision of the article, F – Final approval of the article.

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derivatives8 or metal-based NPs. Meanwhile, the most studied
metal-based NPs are gold, platinum, iron oxide, and
bismuth.4,12-18 Alternatively, natural chemicals are also being
explored as radiosensitizers such as c-phycocyanin,
piperlongumine, and Oroxylum indicum (OI) plant.19-21
The application of radiosensitizer in combination with
radiotherapy using proton beam might induce more cancer cell
death via the production of reactive oxygen species (ROS).
Intracellular ROS is an essential factor in the mechanism of cells
death, especially in cancer radiotherapy. ROS usually exist in
normal healthy cells, but a higher level of ROS would be
produced in cancer cells,22 especially during radiotherapy.23
Higher ROS levels indicated the higher oxidative stress occurred
in the cells, which might involve several mechanisms such as
membrane damages, lipid peroxidation, and DNA damages that
lead to cancer cells death.22 The cancer cells might undergo
apoptosis or programmed cell death when the intracellular ROS
were stimulated excessively.24
Our previous work had evaluated the effect of bismuth oxide
NPs (BiONPs) on ROS for proton beam, which produced the
highest ROS level compared to gold NPs, superparamagnetic
iron oxide NPs and platinum NPs.4 Besides, the combination of
BiONPs with the cisplatin (Cis) as well as baicalein-rich fraction
(BRF) from IO plant leaves extracts had also been investigated,
but for different clinical radiotherapy beams such as HDR
brachytherapy,14,25,26 6 MV of photon beam, and 6 MeV of an
electron beam.15,27
Following this work, the present study is the first to investigate
the influence of BiONPs, Cis, and BRF, individually or in
combinations, on the ROS generation in proton beam irradiated
human colon carcinoma cells.
Materials and Methods
Preparation of the BiONPs, Cis, and BRF
The BiONPs used in this experiment were 60 nm in size and rod-
shaped. The BiONPs were synthesized using hydrothermal
methods and characterized, as reported in previous literature.28,29
The 0.5 mM of BiONPs would be freshly prepared by dilution
with the McCoy's 5A medium (Gibco, Life Technologies, UK)
and mixed before being used. The Cis powder (Tokyo Chemical
Industry) was diluted with dimethyl sulfoxide (DMSO) to a final
concentration of 1.30 µM.30 Meanwhile, the baicalein-rich
fraction (BRF) was extracted and fractionated from OI
leaves.25,31 The BRF powder would be dissolved in DMSO to a
final concentration of 0.76 µg/ml, based on the previous
cytotoxicity study.32
Technologies, South America) and 1% penicillin-streptomycin)
(Gibco, Life Technologies, USA) were used to culture the cells.
The cells were detached and sub-cultured using 0.25% Trypsin-
EDTA (Gibco, Life Technologies, USA). The cells would be
reseeded into 96 wells plates for the ROS measurement study
with the treatments of the BiONPs, Cis, BRF, and their
combinations, as presented in Table 1.
Table 1. Treatment components for the ROS measurement study.
Treatment Components Details
Irradiation 0 Gy (No irradiation), 3 Gy, 6 Gy
BiONPs 0.5 mM
Cis 1.30 µM
BRF 0.76 µg/ml
BC BiONPs + Cis
BB BiONPs + BRF
BCB BiONPs + Cis + BRF
Cell Irradiation Set Up
The irradiation was conducted using a proton particle beam
accelerator (Mitsubishi Electric, Hyogo, Japan) at Hyogo Ion
Beam Medical Centre, Japan. The proton beam field size was set
at 20 cm × 20 cm and the source to surface distance (SSD) of
100 cm. To ensure that the samples got the highest dosage
possible, 6 cm of polyethylene-based solid water phantom slabs
(Taisei Medical, Osaka, Japan) were put on top of plates as a
buildup medium, considering the Spread-out Bragg Peak
(SOBP) of 6 cm. The residual range is within 3% of the SOBP.33
The cells in the 96 wells plate were positioned positions inside
the SOBP area between the phantom slabs, as shown in
Figure 1. The cell samples were exposed to 3 and 6 Gy of 150
MeV proton beams at the dose rate of 0.1 Gy/s in a single
fraction. The set up follows our previous works.4,14,34

Cell Culture
Figure 1. Set up for the proton beam therapy irradiation.
Human colon carcinoma cells (HCT 116) were used in this
study. The McCoy's 5A medium (Gibco, Life Technologies,
UK), supplemented with 10% fetal bovine serum (Gibco, Life

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Effective Atomic Number (Zeff) Calculation

The Zeff of each of the individual components (BiONPs, Cis, and
BRF) were calculated using Equation 1:

𝑍𝑒𝑓𝑓 = 𝑚√∑ 𝑓𝑖 𝑍𝑖𝑚 Eq. 1

where f is the fraction of each element to the total electron
number in each composite, and m is the exponent of 2.94. 35 The
estimated Zeff of each component were used as references to
obtain the mass energy-absorption coefficient for each
compound from the physical data references of the National
Institute of Standard and Technology database.36 The calculated
Zeff is stated in Table 2.

Table 2. The calculated Zeff for individual treatment components.

Treatment Components Calculated Zeff

BiONPs 78 (≈ Platinum)30
Cis 62 (≈ Samarium)30
BRF 6 (≈ Carbon)
Note: Zeff of BiONPs and Cis had been mentioned in our previous published
works.30

Statistical Data
Data are presented as the means ± standard error of means
(SEM) of at least three independent experiments using
OriginPro 2019 software (OriginLab Corporation, US). Two-
way analysis of variance (ANOVA) and Bonferroni Post Hoc
test with the significance level (p) of 0.05 were used in this
study.

Results
Figure 2a shows the ROS measurements of cells treated with
individual treatment components, consisting of BiONPs, BRF,
and Cis, while Figure 2b indicates the ROS readings of cells
treated with the combination treatments of BB, BC, and BCB.
The values were compared before the cells were irradiated, the
non-irradiated cells (0 Gy) and the irradiated cells with 3 Gy
dose of the proton beam.
Overall, the highest ROS measured after irradiation for
individual treatment was marked by Cis treatment, followed by
BiONPs. The ROS after Cis treatment was significantly
different compared to the control cells (p-value<0.05). However,
ROS production by the cells after being treated by BRF was
lower than ROS in the control cells. Nevertheless, the difference
of ROS levels when BRF were compared to BiONPs or Cis were
significant (p-value<0.05).
In addition, ROS induced by the combination treatments were
dominated by BC treatment; meanwhile, the ROS levels induced
after BB and BCB treatments were lower than the ROS in the
control cells (p-value>0.05). In comparison, the intracellular
ROS after BB and BC treatments were significantly different (p-
value<0.05).
Figure 2. Intracellular ROS measurements for each (a) individual
treatment component and (b) combination treatment component.
From the previous result, Cis and BC treatments were further
analyzed for their ROS production on time and the radiation
doses. Figure 3 shows the increment of ROS production as time
passed. The ROS levels before the cells were irradiated and after
irradiations, regardless of the time periods, were significantly
different (p-value<0.05). However, it was revealed that the
intracellular ROS after Cis treatment induced higher ROS than
BC combinations. This circumstance can also be seen in
Figure 4, in which the intracellular ROS increased as the
radiation doses increased, but the difference among the
irradiation doses given was not significant (p-value>0.05). The
intracellular ROS before irradiations were significantly different
from the ROS after irradiation periods (p-value<0.05).

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Figure 3. Generation of intracellular ROS of Cis- and BC-treated Figure 4. Generation of intracellular ROS of Cis- and BC-treated
cells over the 4 hours after irradiation. cells after 2 hours irradiation of different doses.

Discussion the ROS levels in the cells treated with BB and BCB
combinations.
Data shows that the presence of Cis could generate the highest
Furthermore, the highest ROS induction from each individual
intracellular ROS compared to other treatments. These present
and combination treatment (Cis and BC treatments) were chosen
results are similar to our previous findings, in which Cis also
and analyzed for their effects over time and irradiation doses.
induced the highest ROS compared to BiONPs and BRF when
The after-irradiation intracellular ROS was first to read 5 hours
the treated cells were irradiated with iridium-192 (192Ir), photon
after the control reading, followed by a one-hour interval. The
beam, and electron beam.27 It could be inferred that the Cis is a
selected parameter was limited to the 3 Gy irradiation dose only.
potent pro-oxidant regardless of the irradiation beam used. Cis
Both Cis- and BC-treated cells generated the increment of ROS
is a commercial clinical drug and is expected to induce a high
in the 2-, 3-, and 4-hours after the irradiation. Figueroa et al.
volume of ROS that could eliminate cancer cells. Cis could
determined that the DCFH2DA agent were stable and could be
increase the ROS level due to the suppression of SESN1
read for 6 hours.42 According to our previous reports, Cis and
proteins.37
BC-treated cells also generated increasing intracellular ROS in
Meanwhile, the BiONPs treatment would produce
breast cancer cells at 3 and 24 hours after irradiation of clinical
intermediate effects of ROS induction. BiONPs prompted lower
beams of 192Ir, photons, and electrons.32
intracellular ROS than Cis, which may be due to the metallic
In addition, Cis- and BC-induced intracellular ROS were also
element of the components. Cis is a platinum-based drug, while
analyzed for the effects of different irradiation doses, which
BiONPs is a bismuth-based complex. A review mentioned that
were 3 Gy and 6 Gy, at the first reading of 2-hours after
Cis could produce many adverse effects compared to bismuth,
irradiation time. Higher irradiation doses would induce higher
and bismuth could reduce the effects.38 The side effects of Cis
intracellular ROS. The dose-dependent ROS induction after
may be influenced by the initiation of the intracellular ROS, and
proton irradiation had also been reported in research that used
the reduction of the effects could be seen in the BC combination.
platinum NPs.14 These results correspond to our previous
Although BC-treated cells induce the highest intracellular ROS
findings in which 3- and 6-Gy doses of 192Ir, photon, and
among the combination treatment components, the presence of
electron beams yielded increasing intracellular ROS. 27 The data
BC causes a lower ROS amount than Cis alone. This condition
in the present study were also in parallel to a report on ROS
occurred maybe due to the radical scavenging and pro-oxidant
detection by Coulomb nanoradiator, which revealed that ROS
properties of bismuth NPs.39
was influenced by the proton irradiation dose and the Zeff
In contrast, compounds obtained from plants had been shown
values.43 However, the current study had estimated that the
to have antioxidant and radioprotective properties. 40 In this
highest Zeff value is for BiONPs, followed by Cis and BRF, as in
study, BRF-treated cells produced lower intracellular ROS
Table 1. Although the Zeff value for BiONPs is comparable to
compared to control cells. The BRF might activate a reduction-
platinum, the intracellular ROS induced in the Cis-treated cells
oxidation reaction in the cells and increase the oxidative stress
is higher. This occurrence could happen due to 35% of other
tolerance level of the cells.41 This condition might further reduce
compounds in the Cis mixed with the platinum (65%), 44 which

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Noor Nabilah Talik Sisin et al: BCB on HCT-116 cells for Proton Beam Pol J Med Phys Eng 2022;28(1):30-36

could lower the Zeff values but combine to generate more Data availability
intracellular ROS.
Data of this research are available. Any enquiries can be
The intracellular ROS levels also could suggest cellular
forwarded to the corresponding author at the email address
damages. A study of proton irradiation on glioblastoma
wandiana@usm.my.
multiforme indicated that intracellular ROS could influence
DNA damages and cell apoptosis.5 Rashid and colleagues
verified that ROS production after the HCT-116 cells underwent Conflicts of interest
proton beam irradiation in the presence of several NPs was Authors declare that there is no conflict of interest regarding the
correlated to the cell survival reduction.4 A later study by Khairil publication of this paper.
Anuar and co-workers also had related ROS induction due to the
presence of platinum NPs to the HCT-116 cells survival.14 Our Funding statement
prior cell survival studies for various clinical radiation beams
showed that BC combinations might be regarded as a safe Ministry of Higher Education Malaysia for Fundamental
therapeutic strategy to healthy normal tissue, only impacting Research Grant Scheme with Project Code:
cancer cells.27,30 Therefore, Cis and BC combinations also have (FRGS/1/2020/STG07/USM/02/2).
the potential to be excellent radiosensitizers, as their presence
could reduce the needed radiation doses of the proton beam Acknowledgments
radiotherapy.
We would like to spread our gratitude toward Hyogo Ion Beam
Medical Center for providing their proton beam facilities for us
Conclusions to conduct this research. Some of the results have been presented
The production of ROS was enhanced in cancer treatment with in the 31st Annual Scientific Congress of Malaysian
the presence of BiONPs and Cis, suggesting the potentials of Oncological Society (ASCOMOS) 2019.
BiONPs, Cis or BC combinations as radiosensitizers for proton
beam therapy. The presence of BRF might be advantageous to
reduce the side effects of the Cis and BiONPs, but it would
reduce the effectiveness of the radiosensitizer combination.

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