Suppression of MnSOD by Andrographolide and its Relation to

Oxidative Stress and Viability of Breast Cancer Stem Cells Treated

with Repeated Doxorubicin Administration

Angie Tara Rachman

, Ayu Suraduhita

, Resda Akhra Syahrani

, Melva Louisa

Septelia Inawati Wanandi

2,4 e

Master’s Programme in Biomedical Science, Faculty of Medicine, Universitas Indonesia

Molecular Biology and Proteomics Core Facilities, Indonesian Medical Education and Research Institute (IMERI),

Faculty of Medicine, Universitas Indonesia

Department of Pharmacology and Therapeutics, Faculty of Medicine, Universitas Indonesia

Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia

Keywords: Breast cancer stem cells, Doxorubicin, Andrographolide, MnSOD, Oxidative stress

Abstract: Cancer stem cells (CSCs) are chemoresistance and could be a preferential target for reversing resistance.

Doxorubicin is one of the most effective chemotherapy agents, but resistance still occurs. Doxorubicin

generates reactive oxygen species (ROS). The increased specific activity of MnSOD has been suggested as

one possible mechanism for breast cancer stem cells (BCSCs) to maintain stemness and survival. The study

performed to determine the effect of andrographolide on oxidative status and its relation to the viability of

BCSCs given repeated doxorubicin. CD24-/CD44+ BCSCs were grown in DMEM/F12 medium with 0,1µM

doxorubicin for 14 days, then treated with a combination of 0,1µM doxorubicin and 0,285 mM

andrographolide until day 22. MnSOD activity was suppressed on day 4 when BCSCs were still sensitive to

doxorubicin treatment. Viability significantly increased on day 20 along with increased MnSOD activity.

Andrographolide restored the sensitivity of BCSCs to doxorubicin, which correlated with MnSOD activity

but not catalase. There was no change in MDA levels in all days of treatment which means BCSCs can

maintain oxidative stress level. Andrographolide supplementation can decrease MnSOD activity but not

catalase and is closely related to decreased cell viability with low sensitivity to doxorubicin.

1 INTRODUCTION

Breast cancer is the most frequently identified cancer

and the leading cause of death from cancer in women

worldwide. (Bray et al., 2018) There are several

treatment options to cure breast cancer, including

mastectomy, chemotherapy, hormone therapy,

radiotherapy, and other therapies. (Sterba et al., 2013)

An anthracycline, doxorubicin, is considered one of

the most effective and most used chemotherapy

agents to treat breast cancer and several other cancer

types. However, resistance to chemotherapy still

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occurs both intrinsically and resistance that develops

during treatment. (Austreid et al., 2014) One of the

causes of doxorubicin resistance is the reduced

amount of the accumulated drug in the nucleus

resulting in a decrease in DNA damage. Failure to

accumulate drugs is caused by active efflux through

the ATP binding cassette (ABC) transporter family.

The upregulation of this transporter is related to drug

resistance. (Shiraga et al., 2001)

Cancer stem cells (CSCs) represent a

subpopulation of cancer cells with close

characteristics to normal stem cells, namely

pluripotency, self-renewal ability, and differentiation.

Rachman, A., Suraduhita, A., Syahrani, R., Louisa, M. and Wanandi, S.

Suppression of MnSOD by Andrographolide and its Relation to Oxidative Stress and Viability of Breast Cancer Stem Cells Treated with Repeated Doxorubicin Administration.

DOI: 10.5220/0010487200280034

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 28-34

ISBN: 978-989-758-499-2

CSCs can originate from normal cells/progenitors

with mutations or environmental changes and can

also come from normal somatic cells that experience

genetic changes. High CSCs indicate a poor

prognosis because cancer stem cells have a slow rate

of division, better DNA repairability, and a lower

ability to experience apoptosis than cancer cells in

general. The causes of cancer stem cells to be more

resistant to cancer therapies such as chemotherapy

and radiation have been proven in vitro studies using

CD24-/CD44+ cancer stem cells. (Phillips et al.,

2006)he

Oxidative stress is a phenomenon that appears

when an imbalance occurs between reactive oxygen

species (ROS) formation and the capability of cells to

detoxify them. The lipid peroxidation is one of the

results of oxidative stress, which will lead to

malondialdehyde (MDA) formation. MDA is one of

the most reliable markers to determine oxidative

stress. (Giera et al., 2012) Oxidative stress is

remarkably known to play an important role in

damaging lipids, protein, and DNA molecule, alter

signalling pathways and impact cancer progressions.

(Lee et al., 2017) ROS also has a role in stem cell

renewal and differentiation. Breast cancer stem cells

can maintain lower ROS levels than differentiated

cells by increasing antioxidant capacity. The

condition can be an obstacle for treatments that use

oxidative stress like chemotherapy and radiotherapy.

(Gorrini et al., 2013) The conditions doxorubicin was

known to generate ROS, although the other

mechanisms, namely through intercalation of DNA

and poisoning topoisomerase II, constitute their

cytotoxic actions. (Lazo et al., 1998)

Previous studies in our laboratory showed that

doxorubicin could reduce the viability of CD24-

/CD44+ breast cancer stem cells (BCSC) at initial

exposure of doxorubicin MnSOD levels were

maintained, thus high ROS levels. After more

extended doxorubicin administration, the viability of

BCSCs increased, and the MnSOD activity was

significantly high, which caused the ROS levels to

decline. Breast cancer stem cells CD24-/CD44+

maintain survival by increasing the activity of the

MnSOD. (Syahrani et al., 2019)

Andrographolide (ANDRO) is a diterpene lactone

derived from the extract of the plant bitter

(Andrographis paniculata), and it has long been

known for its antioxidant (Xu et a;., 2019), anticancer

(Siripong et al., 1992), immunostimulatory (Puri et

al., 1993), anti-inflammatory (Abu-Ghefreh et al.,

2009), and anti-viral (Manjula et al., 2018) activities.

Andrographolide has demonstrated in promoting

apoptosis in BCSCs by inhibition of anti-apoptotic

protein survivin. (Yunita et al., 2017) Initially,

andrographolide was reported to be a ROS scavenger

(Xu et al., 2019), but natural compounds may

demonstrate both antioxidant and prooxidant

characteristics that depend on the concentrations and

exposure. (Sznarkowska et al., 2017) In a parallel

study, it showed that the administration of

andrographolide could cause cell death in BCSCs.

The study performed to determine the possibility of

andrographolide re-sensitising doxorubicin resistance

due to increased MnSOD activity.

2 MATERIALS AND METHODS

2.1 Cell culture

Breast cancer stem cells CD24-/CD44+ were

obtained from the Department of Biochemistry and

Molecular Biology, Faculty of Medicine Universitas

Indonesia and grown in serum-free Dulbecco’s

Modified Eagle Medium / Nutrient Mixture F-12

(DMEM/F-12 medium) (Gibco, Thermo Fisher

Scientific, Inc. Waltham, MA, USA) supplemented

with 1% penicillin-streptomycin (Gibco, Thermo

Fisher Scientific, Inc. Waltham, MA, USA) and

amphotericin B (Gibco, Thermo Fisher Scientific,

Inc. Waltham, MA, USA). BCSCs were maintained

in 5% CO2 at 37°C. Cells seeded at 1 x 10

cells/well

in 12 well plates.

2.2 Doxorubicin and Andrographolide

Treatment

Doxorubicin (Sigma-Aldrich, St. Louis, Missouri,

USA) was prepared in serum-free DMEM/F12

medium at a final concentration of 0,1µM.

Andrographolide prepared in DMSO (Sigma-

Aldrich, St. Louis, Missouri, USA) and diluted in

serum-free DMEM/F-12 medium at a final

concentration of 0,285 mM. Cells were treated with

0,1 µM doxorubicin every two days. After 14 days of

treatment, the cells were treated with a combination

of doxorubicin and andrographolide until day 22.

Counting Cells used by Trypan blue exclusion assay.

Cell viability was determined by dividing the number

of living cells of the treatment group to the number of

living cells of the control group.s

2.3 Protein Isolation

According to the manufacturer's protocol, RIPA

buffer (Thermo Fisher Scientific, Illinois, USA) was

Suppression of MnSOD by Andrographolide and its Relation to Oxidative Stress and Viability of Breast Cancer Stem Cells Treated with

Repeated Doxorubicin Administration

used to isolate protein from BCSC CD24-/CD44+.

The supernatant containing protein stored in a new

tube at -80

2.4 MnSOD Activity

MnSOD activity of protein samples was determined

using the RANSOD kit (Randox Lab, Crumlin, UK).

Protein samples used for the assay of SOD. To inhibit

Cu / ZnSOD, 5mM sodium cyanide was added.

Absorbance measured at wavelengths of 505 nm on a

spectrophotometer for 30 seconds. Preparation of

percentage inhibition versus standard/protein sample

assay and standard inhibition curve following the

recommended kit manual. The SOD activity values

for protein samples were read based on a curve

divided by the protein concentration.

2.5 Catalase Activity

50 μL of standard/sample were added into a tube

containing 950 μL of H

. The homogenised mixture

read by absorbance at 210 nm. The absorbance

observations were carried out at the first 30 seconds

(00:30) and two minutes after that (02:30) using a

stopwatch. The catalase activity was measured using

the catalase standard curve made in several dilutions,

divided by the protein concentration.

2.6 MDA Levels

The concentration of MDA (malondialdehyde)

formed from lipid peroxidation was measured using

the Wills method. The principle is that MDA will

react with thiobarbituric acid (TBA) at a temperature

of 90

C-100

C in an acidic atmosphere to form a pink

compound that provides maximum absorbance at a

wavelength of 530 nm. The concentration is

calculated based on the calculations obtained from the

linear regression of the MDA standard curve. The

MDA concentration divided by the protein

concentration.

2.7 Statistical Analysis

The data were analysed with IBM SPSS Statistics 22.

The average probability analysed with a Shapiro-

Wilk test. The data performed as a mean value ±

standard deviation (SD). Statistical evaluation of the

significant differences performed using variance

(ANOVA); the LSD/Tukey test used for multiple

comparisons. The significance level was at p<0.05.

3 RESULTS

3.1 Measurement of MnSOD Specific

Activity

The activity of antioxidant enzyme MnSOD in

doxorubicin treated BCSCs and a combination of

doxorubicin and andrographolide treatment after day

14 to day 22 were assayed. As shown in Figure 1,

BCSCs’ MnSOD activity significantly decreased

compared to control with no treatment on day 4 (p <

0.05). There was no statistical difference between the

MnSOD activity of doxorubicin treated BCSCs

compared to the control group on day 14.

Interestingly, there was a significant increase of

MnSOD activity on day 20 of doxorubicin-induced

BCSCs compared to control on the same day (p <

0.05) and also compared to day 14 of doxorubicin-

induced BCSCs (p < 0.05). Andrographolide was

given on the 14th day and reduced the MnSOD

activity of doxorubicin-induced BCSCs significantly

(p < 0.01).

Figure 1: Effect of doxorubicin and doxorubicin-

andrographolide on BCSCs MnSOD specific activity at

specified times. MnSOD activity was determined using the

RANSOD kit (Randox Lab, Crumlin, UK). Statistical

analysis conducted as described in Materials and Methods.

Significant differences from control values (*, p < 0.05)

(**, p < 0.01) are indicated by the asterisks.

3.2 Measurement of Catalase Specific

Activity

Catalase specific activity was measured based on a

standard H

decomposition curve by measuring the

absorbance of H

with a blank at a concentration of

1: 1000; 1: 2000; 1: 4000; 1: 8000; 1: 16000; 1: 32000

and 1: 64000. The absorbance observations at 210 nm

were carried out at the first 30 seconds (00:30) and

two minutes after that (02:30) using a stopwatch.

Catalase activity compared in doxorubicin treated

BCSCs, doxorubicin-andrographolide treated BCSCs

and no treatment control. There was no difference

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

between the catalase activity of doxorubicin-induced

and control BCSCs on day 4 and day 14. There was a

significant decline of doxorubicin-induced BCSCs on

day 20 compared to a control of the same day (p <

0.05). No significant differences observed between

the catalase activity of andrographolide

administration on doxorubicin-induced BCSCs' and

doxorubicin-induced BCSCs.

Figure 2: Effect of doxorubicin and doxorubicin-

andrographolide on BCSCs catalase specific activity at

specified times. Catalase activity was determined using the

catalase standard curve. Statistical analysis conducted as

described in Materials and Methods. Significant differences

from control values (*, p < 0.05) (**, p < 0.01) are indicated

by the asterisks.

3.3 MDA Levels

Oxidative stress is one of the factors that contribute

to the cytotoxicity of doxorubicin. The measurement

of the peroxidation product levels – malondialdehyde

(MDA) was used to calculate the lipid peroxidation

status in BCSCs. Lipid peroxidation was assayed by

the thiobarbituric acid method. As shown in Figure 3,

the MDA levels of BCSCs exposed to doxorubicin

show no difference compared to control.

Andrographolide administration showed a trend of

increased MDA levels on doxorubicin-induced

BCSCs, but the proportion was not significant.

Figure 3: Effect of doxorubicin and doxorubicin-

andrographolide on MDA levels at specified times.

Statistical analysis conducted as described in Materials and

Methods. Significant differences from control values (*, p

< 0.05) (**, p < 0.01) are indicated by the asterisks.

4 DISCUSSION

Doxorubicin is one of the most effective and most

used chemotherapy agents to treat breast cancer and

several other cancer types. One of how doxorubicin

causes cell death is through the mechanism of ROS

formation, increasing oxidative stress. Increasing free

radicals will cause oxidative stress. (Xu et al., 2005)

Syahrani et al. demonstrated that breast cancer stem

cells' sensitivity to doxorubicin decreased when given

long-term exposure. Breast cancer stem cells

(BCSCs) can maintain stemness and survival through

increased antioxidant enzyme MnSOD activity.

(Syahrani et al., 2019)

In a parallel study at the same laboratory,

doxorubicin reduced CD24-/CD44+ breast cancer

stem cells' viability until day ten. The viability

increased on the 14th day, so it indicated the time

when the BCSCs sensitivity decreases. The BCSCs in

this study could not be declared resistant in a study

conducted by Lukyanova et al., a doxorubicin-

resistant variant of breast cancer cells was obtained

by growing cells medium containing doxorubicin

with stratified concentrations over a more extended

period. (Lukyanova et al., 2009) The viability of

CD24-/CD44+ BCSCs continued to increase up to

74.19% on the 20th day. The administration of

andrographolide carried out every other day on day

14 until day 18 caused a decrease in the viability of

breast cancer stem cells exposed to doxorubicin. This

result indicates that andrographolide inhibits the

growth of BCSCs that had reduced sensitivity to

doxorubicin.

Figure 4: Effects of androgapholide on the viability of

doxorubicin-induced BCSCs. BCSCs were treated with 0,1

µM doxorubicin until day 14 then, doxorubicin treated

BCSCS were supplemented with 0,285 mM

andrographolide.

Suppression of MnSOD by Andrographolide and its Relation to Oxidative Stress and Viability of Breast Cancer Stem Cells Treated with

Repeated Doxorubicin Administration

CSCs from some types of tumours had lower ROS

levels than differentiated cells. Its indicated that

CSCs could maintain a low ROS level, which might

help them protect themselves from damages caused

by ROS. This low level of ROS was partly caused by

an increase in the production of ROS scavenger

enzymes. (Diehn et al., 2009) Therefore, we analysed

the activity of the antioxidant enzyme. MnSOD was

the most potent antioxidant enzyme because it acts as

the first detoxification enzyme needed to protect cells

from ROS's toxicity produced by metabolism. This

enzyme catalysed the dismutation of potentially

dangerous superoxide anions into hydrogen peroxide

and molecular oxygen. (Oberley, 2005).

Increased expression of MnSOD protein could

lead to resistance to therapy. The mechanism by

which MnSOD protects cells from oxidative damages

was thought by maintaining mitochondrial function

and preventing the reduction in ATP synthesis caused

by oxidants. (Suresh et al., 2003) In the MnSOD

activity analysis using the RanSOD kit, the results of

MnSOD activity from CD24-/CD44+ BCSCs

exposed to doxorubicin on day four significantly

lower compared to the control without treatment. It is

caused by the changes in the balance of oxidative

stress levels, which is one of the doxorubicin's

mechanisms of action. The imbalance between high

levels of oxidative stress and antioxidants could cause

mutations in DNA and damage to genes that produce

antioxidant proteins. (Bagchi et al., 1998) The result

was that BCSCs lose their ability to fight free radicals

due to disruption in antioxidant proteins' production.

Also, MnSOD activity has been widely used to

combat the high amount of ROS, so that the measured

activity was reduced. On day 14, the MnSOD activity

of CD24-/CD44+ BCSCs exposed to doxorubicin

showed no significant difference compared to control.

On day 20, the MnSOD activity of doxorubicin-

induced BCSCs was significantly higher than the

control and the doxorubicin-induced BCSCs on day

4. This research aims to clarify the role of MnSOD in

causing resistance to doxorubicin in BCSCs by

supplementation of androgapholide. The suppression

of MnSOD by andrographolide affected the viability,

sensitising BCSCs to doxorubicin treatment. Another

important finding is that the MDA level after

andrographolide exposure showed a higher trend

compared to doxorubicin alone, although not

significant, the result suggested that there was an

increase in oxidative stress. We suggested that

andrographolide could reduce MnSOD activity and

revive the oxidative stress induced by doxorubicin, so

that viability decreases.

Catalase gives protection against the deleterious

effect against H

and decomposes them to oxygen

and water. Catalase is located in the peroxisomes.

(Kirkman et al., 2007) On day 4 and 14, BCSCs was

sensitised to doxorubicin treatment, and the catalase

level was no different compared to the control

without treatment. Catalase is known to have an

essential role in developing tolerance to oxidative

stress in cells, especially when there is limited

glutathione and decreased GPx activity (Wassmann et

al, 2004) or increasing H

levels. (Yamada et al.,

1991) On day 20, the catalase activity of doxorubicin-

induced BCSCS was decreased significantly

compared to day 14 doxorubicin-induced BCSCS and

control of the same day, but the MDA level remains

stable. The finding of lower catalase activity in this

day might be explained with the regulation of catalase

expression. The catalase core promoter is highly

conserved, allowing efficient binding of transcription

factors to the DNA binding sites and leading to the

positive or negative regulation of catalase expression.

(Nenoi et al., 2001; Glorieux et al., 2015) Epigenetic

changes have been shown to regulate catalase

expression in acute myelogenous leukaemia cells

resistant to doxorubicin, reducing catalase protein

levels compared to the parental cell lines. (Lee et al.,

2012) Besides, catalase expression is also regulated at

the RNA level and post-translational modifications,

causing decreased catalase activity. (Glorieux et al.,

2015) There was no statistical difference in the

catalase activity between doxorubicin-

andrographolide induced BCSCs and doxorubicin-

induced BCSCs on day 20. Based on our results, we

suggested an increase in MnSOD activity, but not

catalase activity leading to a decrease in viability.

Furthermore, increased expression of MnSOD was

associated with a decrease in viability induced by

oxidative stress.

Oxidative stress is one of the factors that

contribute to the cytotoxicity of doxorubicin. ROS

has a short life span, which is not easy to be detected.

(Sanz, 2016) Measurement of lipid peroxidation end

product – malondialdehyde (MDA) was used to

determine lipid peroxidation as a convenient

biomarker for ROS related damage. On day 4, there

was no statistically significant difference between the

MDA levels of BCSCs induced with doxorubicin and

control. After 14 days of exposure to doxorubicin, the

MDA level of BCSCs induced with doxorubicin

shows a higher trend than the control group.

However, when compared to day 4, MDA

concentration was stable.

Similarly, the MDA level of doxorubicin-induced

BCSCs on day 20 was remained stable at the same

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

range as day 4 and day 14. A state of redox imbalance

between oxidants and antioxidants is a common

hallmark of cancer resistance to treatment. High ROS

exposure was given to increase oxidants within the

cell and cause ROS-mediated damaged biomolecules

such as DNA and protein. (Ziech et al., 2011) We

suggested that the stable MDA concentration between

MDA treated group showed the ability of BCSCs to

maintain homeostasis and evaded cancer cell death by

developing an antioxidant defence. SOD and catalase

are the best enzymatic antioxidants on scavenging

ROS (Banerjee et al., 2017) suggested that they were

effectively scavenged free radicals and kept the

amount of MDA in doxorubicin-induced BCSCs

balanced. After exposure of andrographolide on day

20, there was a trend that the MDA level of

doxorubicin-induced BCSCs was increasing.

Andrographolide may have prooxidant and

antioxidant characteristics. Previous studies have

shown that andrographolide can trigger intracellular

ROS formation, contributing to apoptosis in cancer

cells. (Banerjee et al., 2017) In this study,

andrographolide did not cause a significant increase

in oxidative stress level, but it could suppress the

antioxidant enzyme that scavenges ROS, MnSOD.

Thus, we demonstrated that andrographolide and

doxorubicin synergistically induced cell death by

MnSOD suppression.

5 CONCLUSIONS

Andrographolide repeated treatment to BCSC can

decrease MnSOD activity but not catalase and

oxidative stress and is closely related to decreased

cell viability with low sensitivity to doxorubicin.

ACKNOWLEDGEMENTS

This research was supported by a grant from the

Penelitian Dasar Unggulan Perguruan Tinggi

(PDUPT).

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JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

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