Advance Publication

Original Article

The cytotoxicity of cyclophosphamide isenhanced in combination with monascus
pigment
Hiromi Kurokawa,1,* Atsushi Taninaka,2 Hidemi Shigekawa,2 and Hirofumi Matsui1,31
Algae Biomass Research and Development, 2Graduate School of Comprehensive Human Sciences, and 3Faculty of Medicine, University of Tsukuba,
1-1-1 Tennodai, Tsukuba 305-8575, Japan(Received 7 December, 2020; Accepted 24 January, 2021)

Monascus pigment is derived from red-mold rice fermented bymonascus purpureus and utilized as a natural coloring agent andnatural food additive in East Asia. Monascus pigment works as aradical scavenger. Some antioxidant combine cancer chemo‐
therapy to protect normal tissue because chemotherapy induceside effect for normal tissue. This combination therapy can
attenuate the cytotoxicity of anticancer drugs by antioxidants
effects. However, the effect of this combination therapy forcancer cells dose not investigate enough. In this study, weinvestigated the combination effect of antioxidants and anti‐
cancer drugs. We selected an antioxidant as monascus pigment
and following four anticancer drugs: doxorubicin, tamoxifen,
paclitaxicel, and cyclophosphamide. Combination treatment withmonascus pigment and cyclophosphamide enhanced thecytotoxicity of cyclophosphamide. Moreover, this combination
treatment accelerated apoptosis. The spot on TLC assay board of
the monascus pigment and cyclophosphamide mixture is different
from the spot of monascus pigment alone and cyclophosphamide
alone. The interaction between monascus pigment and cyclo‐
phosphamide can produce some cytotoxicity compounds oraccelerate intracellular cyclophosphamide accumulation. Hence,
we concluded that the interaction of both cyclophosphamide andmonascus pigment involved enhancement of cyclophosphamide
cytotoxicity.
Key Words:

Monascus purpureus, cyclophosphamide, apoptosis,
BcL-2, Bax

Monascus pigment (MP) is derived from red-mold rice
fermented by Monascus purpureus and utilized as a natural
coloring agent and natural food additive in East Asia.(1,2) The
components of MP have been identified. Monacolin K inhibit the
enzyme 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA)reductase, which catalyzes the reduction of HMG-CoA to meval‐onate.(3) The same substance approved by the US Food and Drug
Administration in 1987 as lovastatin.(4) γ-Aminobutyric acid anddimerumic acid possessd antihypertensive effects and antioxidant
activities.(5–7) Ankaflavin and monaphilone A inhibited tumor
growth by induce of apoptosis or anti-inflammatory effect.(8)Cancer patients were estimated at 18.1 million new casesand 9.6 million deaths by the World Health Organization in
2018.(9) Chemotherapy is one of the common treatment of cancer.
Alkylating agents bind covalently with DNA, crosslink themand induce DNA strand breaks. Topoisomerase inhibitors prevent
DNA replication.(10) Microtubule targeting agents interrupt
mitotic spindles and arrest mitosis.(11) However, many traditional
chemotherapy drugs have nonspecific distribution and uncontrol‐
lable release of drugs. Thus, side effect for normal tissue isdoi: 10.3164/jcbn.20-197

serious problem in chemotherapy. To inhibit the side effect ofanticancer drugs, it is investigated that combination therapy with
anticancer drugs and antioxidants.(12–15) Injury for normal tissue
attenuate by this combination therapy. However, the effect of thiscombination therapy for cancer cells dose not investigate enough.
In this study, we investigated the combination effect of anti‐oxidants and anticancer drugs. We select MP as an antioxidant.
We previously reported that MP scavenge reactive oxygen
species (ROS), especially mitochondrial ROS (mitROS). The
activation of acid ceramidase was attenuated because of mitROS
reducing by MP. Acid ceramidase degrades ceramides to sphin‐
gosine and fatty acids. Increase of ceramide accumulation induce
apoptosis. From these results, cancer specific apoptosis was
induced by MP.(16) These results occurred by directly antioxidant
effect. In this study, we evaluated indirectly antioxidant effect for
anticancer drugs.
Materials and Methods
Cell culture. The murine 4T1 mammary carcinoma cell line
was purchased from RIKEN CELLBANK. The 4T1 cells were
cultured in high-glucose RPMI1640 (Wako Pure Chem Ind. Ltd.,
Osaka, Japan). The culture medium contained 10% of inactivatedfatal bovine serum (Biowest LLC, Kansas City, MO) and 1% of
penicillin/streptomycin (Wako Pure Chem). The cell was cultured
in 5% CO2 cell culture incubator at 37°C.
Cell viability assay. Cell viability was measured using the
Cell Counting Kit-8 according to manufacturer’s protocol.(17) 4T1
cells were cultured on 96-well plates at 5 × 103 cells/well andincubated overnight. The supernatant was aspirated and the
medium was replaced. To determine the cytotoxicity of MP(Wako Pure Chem), these media contained 0, 50, 100, 150, 200,
300, and 400 μg/ml of MP respectively. Cells were incubated at
37°C for 24 h. After incubation, the supernatant was aspirated
and then incubated with medium containing 10% Cell Counting
Kit-8. The absorbance at 450 nm was measured by a DTX880
multi-mode microplate reader (Beckman Coulter, Brea, CA).
The effect of combination therapy of MP and anticancer drugs
was evaluated. Doxorubicin (DOX), tamoxifen (TAM), pacli‐
taxicel (PTA) and cyclophosphamide (CPA) (Wako Pure Chem)
were selected as anticancer drugs. 4T1 cells were cultured on 96well plates at 5 × 103 cells/well and incubated overnight. The
supernatant was aspirated and the medium containing MP and
anticancer drugs was replaced. Cells were incubated at 37°C for
24 h. After incubation, the supernatant was aspirated and then
*To whom correspondence should be addressed.
E-mail: hkurokawa.tt@md.tsukuba.ac.jp

J. Clin. Biochem. Nutr. | Published online: 7 April 2021 | 1–6

Detection of apoptotic cells by Hoechst 332342 staining.

The apoptosis of 4T1 cells was detected by the Hoechst 33342.
4T1 cells were cultured on 6-well plates at 5 × 104 cells/well andincubated overnight. The supernatant was aspirated and incu‐
bated with or without 200 μg/ml MP containing 5 mg/ml CPA
for 24 h. The supernatant was thereafter aspirated and then incu‐
bated with MSF buffer containing 10 μM Hoechst 33342 for 10
min. Then cells were rinse with MSF three times and added MSF.
Apoptosis, with condensed and fragmented nuclei, was analyzed
using a fluorescence microscope (IX83, Olympus Inc., Tokyo,
Japan). Fluorescence intensity of Hoechst33342 was excited at
340–390 nm filter and the emission was collected at 420 nm
filter.
TLC assay. CPA, MP and MP with CPA solution were

Results
MP act as a radical scavenger. The effect for 4T1 cells was
examined. Cells were incubated for 24 h in medium containing
MP. MP had no effect in cell viability for 4T1 until 200 μg/ml.
However cytotoxicity of MP occurred in dose-dependent mannerfrom 300 μg/ml (Fig. 1). Intracellular ROS production wasmeasured using ESR. The signal of DMPO was gradually low in
dose-dependent manner (Fig. 2A). Especially, the DMPO signal
in 200 μg/ml MP was significantly decreased compared with in 0μg/ml MP (Fig. 2B).
MP enhanced the cytotoxicity of CPA. The combination
effect with anticancer drugs and MP was examined. 200 μg/ml
MP did not showed cytotoxicity for 4T1 and acted as a radical
scavenger, thereby we chose 200 μg/ml MP for investigatingsynergy effects with each anticancer drugs. The cytotoxicity of
DOX, PTX and TAM did not enhance by MP (Fig. 3A–C). The
cell viability of 5 mg/ml CPA without MP was 59.6 ± 7.1%,
while it with MP was 32.4 ± 3.3% (Fig. 3D). The cytotoxicity of
5 mg/ml CPA was significantly enhanced by MP.

140

**

120
100
80
60
40

0

0

100 µg/ml

200 µg/ml

50

100

200

300

400

MP concentration (µg/ml)
Fig. 1. Cytotoxicity of MP. 4T1 cells were exposed to culture medium
containing several concentrations of MP for 24 h, then WST assay was
performed. Data are expressed as means ± SD (n = 4). **p<0.01.

B
0 µg/ml**

20

Peak intensity/Base intensityAspotted on a silica gel TLC plate, which was developed in 1Propanol: water (8:1, v/v). CPA was detected by ninhydrinethanol TS spray (Wako Pure Chem). TLC plate was imaged by adigital camera.

Cell viability (%)

incubated with medium containing 10% Cell Counting Kit-8. The
absorbance at 450 nm was measured by a DTX880 multi-mode
microplate reader.
Western blot assay. 4T1 cells were incubated overnight in
60-mm dishes. Cells were incubated containing 200 μg/ml MP
and 5 mg/ml CPA medium for 24 h. The cells were rinsed with
PBS three times and RIPA buffer was added in on ice, thenboiling at 70°C for 10 min. These whole cell lysates were used asWestern bolt sample. For SDS-polyacrylamide gel electro‐
phoresis, the samples were added into wells of NuPAGE®
Novex® 4–12% Bis-Tris gels (Thermo Fisher Scientific K.K.,
Kanagawa, Japan). The gels were electrophoresed at 100 V for
40 min, and proteins were transferred onto a PVDF membrane(Bio-Rad Laboratories, Hercules, CA) by electrophoresis at 1.2
mA/cm2 for 60 min. The membrane was blocked for 60 min with
PVDF blocking reagent from the Can Get Signal® (TOYOBO
CO. LTD., Osaka, Japan) and probed with primary and secondary
antibodies. Anti-rabbit B-cell lymphoma 2 (Bcl-2), Bcl2associated X protein (Bax) and p-Akt (ser473) (Cell Signaling
Technology Japan K.K., Tokyo, Japan) antibodies (1:1,000) wereadded to the Can Get Signal® Immunoreaction Enhancer Solution
1 (TOYOBO), and then exposed to the membrane overnight.
After the primary antibody solution was aspirated, the membrane
was washed three times with PBS containing Tween 20. The
secondary HRP-linked anti-rabbit IgG antibody (Cell Signaling
Technology Japan K.K.) (1:1,000) was added to the Can Get
Signal® Immunoreaction Enhancer Solution 2 (TOYOBO) and
exposed to the membrane for 60 min. Lumina Forte Western
HRP substrate (Millipore Co., Billerica, MA) was used to visu‐
alize the membrane. Images of the blots were captured on the
FUSION FX7.EDGE (Vilber Lourmat, Co, Torcy, France). βActin (Cell Signaling Technology Japan K.K.) was detected as
the control for protein loading.

*

5
4
3
2
1
0

0

100

200

MP (µg/ml)
Fig. 2. Intracellular ROS production after treatment MP. (A) The electron spin resonance (ESR) spectra. (B) Relative ESR intensity. The signal of
DMPO was gradually low in dose-dependent manner by MP. Data are expressed as means ± SD (n = 3). *p<0.05.

2

doi: 10.3164/jcbn.20-197

MP (–)MP (+)

100
80
60
40
20
0

0

0.1

C 120
Cell viability (%)

Cell viability (%)

A 120

100
80
60

20

**

80

**

60
40
20
0

0

10

100

1

D 120
Cell viability (%)

Cell viability (%)

100

MP (–)MP (+)

0

10

TAM concentration (μg/ml)

DOX concentration (μg/ml)

B 120

**

40

0

1

MP (–)MP (+)

100

MP (–)MP (+)

80

**

60
40
20
0

PTX concentration (µg/ml)

0

1

5

CPA concentration (mg/ml)

Fig. 3. The cytotoxicity of anticancer drugs combination with MP. The combination effect with anticancer drugs and MP was examined. (A) DOX,
(B) PTX, (C) TAM, and (D) CPA. The concentration of MP was 200 μg/ml. Data are expressed as means ± SD (n = 4). **p<0.01.

Combination therapy with MP and CPA decreased the
BcL-2 expression and increased the Bax expression. Cells

were exposed medium containing 5 mg/ml CPA with or without
MP for 24 h. Then the expression of Bcl-2 and Bax wereanalyzed by Western blotting. We observed the combination
therapy with MP and CPA reduction in the levels of the antiapoptotic protein Bcl-2 (Fig. 4A and B). While a concomitant
increase in the expression level of pro-apoptotic protein Bax was
observed (Fig. 4C and D). The p-Akt signaling pathway plays asignificantly role in regulating cell apoptosis.(18) We measured thep-Akt expression. The results showed that p-Akt expression was
decreased in cells treated with CPA and MP (Fig. 4E and F).
These findings indicated that the combination with MP and CPA
alters the expression of proteins involved in the regulation of
apoptosis.
MP induces apoptosis in RGK1 cells. 4T1 cells were treatedwith or without 200 μg/ml MP containing 5 mg/ml CPA for 24h, thereafter cell apoptosis was confirmed by Hoechst 33342
staining assay. Morphological changes indicating cell apoptosis
such as condensation of both chromatin and nuclear fragmenta‐
tions were found in MP treated cells (Fig. 5, white arrows). The
result showed that MP treatment induced cancer cellular apop‐
tosis.
The spot of MP with CPA solution showed different from
MP solution. Thin mark is made at the bottom of the plate toapply the sample spots. Samples solutions are applied on the
spots marked on the line. Then elute the plate using 1-Propanol
and water. CPA spot was detected by ninhydrin reaction (Fig.
6A). The spot of CPA with MP was different from without CPA(Fig. 6B and C). Moreover, the spot of MP alone and MP with
CPA were different at 100, 250, and 300 pixels (Fig. 6D and E).

H. Kurokawa et al.

Discussion
In this study, we demonstrated that the cytotoxicity of CPA
enhanced by MP. The combination treatment with antioxidants
and anticancer drugs attenuated the side effect for normal tissue
by anticancer drugs. However, the investigation of this combina‐
tion treatment for cancer tissue was not enough. This combina‐
tion treatment may also attenuate the cytotoxicity of anticancer
drugs for cancer tissue. In this study, we investigated the cytotox‐
icity of the combination treatment with antioxidant and anti‐
cancer drugs for cancer cell. MP is an antioxidant.(16) We demon‐
strated the cytotoxicity of MP for 4T1 murine breast cancer cells.
The cytotoxicity of MP did not show until 200 μg/ml, however
the cell viability compared with 0 μg/ml was decreased signifi‐
cantly from 300 μg/ml (Fig. 1). We measured the antioxidant
effect of MP. MP act radical scavenger in 200 μg/ml which
concentration did not show cytotoxicity (Fig. 1 and 2). Fromthese results, we selected the MP as an antioxidant.
We investigated the cytotoxicity of combination treatment with
MP and anticancer drugs for cancer cell. DOX, PTX, TAM, and
CPA are used in the standard of care for breast cancer in Japan.
The cell viability of DOX without MP and with MP did notchange (Fig. 3A). The cytotoxicity in 10 ng/ml PTX enhancedby MP, however the cytotoxicity in 100 ng/ml PTX attenuated(Fig. 3B). The cell viability in 10 μg/ml TAM increased by MP(Fig. 3C). The cytotoxicity in 5 mg/ml CPA significantly
enhance with MP (32.4 ± 3.3%) compared without MP (59.6 ±
7.1%). Researchers have been reported that antioxidants canprevent the side effect of anticancer drugs for normal tissue. In
this study, we cleared that the antioxidant can enhance the cyto‐
toxicity of specific anticancer drugs.
We measured the expression of BcL-2 and Bax. The BcL-2
regulate the permeabilization of the mitochondrial outer

J. Clin. Biochem. Nutr. | Published online: 7 April 2021 | 3

C

Control MP

CPA MP+CPA

E

Control MP

BcL2

Bax

p-Akt

β-actinβ-actinβ-actin

D 2.5

1
0.5

0.2
0

A
CP

CP
P+

P+

A

A
CP

M

l
ro
nt

P

M

M

Co

P+

CP

CP

A

A

P
M

nt

ro

l

0

Co

0.4

A

0

0.6

M

0.2

0.8

l

0.4

1.5

1

ro

0.6

p-Akt/β-actin

0.8

*

1.2

2

1

Bax/β-actin

BcL2/β-actin

1.2

F 1.4

*

nt

*

Co

B 1.4

CPA MP+CPACPCPA MP+CPA

PControl MP

M

AFig. 4. Expression of BcL-2, Bax, and p-Akt. BcL-2, Bax, and p-Akt were detected by Western blot. Compared to control, BcL-2 expression was
decreased in combination with MP and CPA (A, B), while Bax expression was increased (C, D). Data are reported as the means ± SD (n = 4).
*p<0.05.

A

B

Fig. 5. Hoechst 332342 staining of 4T1 cells. The cells undergoing apoptosis demonstrated apoptotic chromatin changes: blebbing, fragmentationand condensation under a fluorescence microscope. Cells were stained (A) without MP or (B) with 200 μg/ml MP containing 5 mg/ml CPA for 24 h.
Data were obtained in three independent experiments.

membrane and suppress apoptosis. Pro-apoptotic elements likeBax promote apoptosis.(19) Compared with control, BcL-2 expres‐sion of the combination treatment with MP and CPA significantly
decreased (Fig. 4A and B), while Bax expression was signifi‐
cantly increased (Fig. 4C and D). The p-Akt regulate BcL-2 andthis expression was decreased the same manner of BcL-2 expres‐sion (Fig. 4E and F). Thus, we considered that the apoptosis by
CPA and MP treatment was induced p-Akt signal pathway. Actu‐
ally, cells treated with MP and CPA were induced apoptosis
compared with CPA alone (Fig. 5). Antioxidants have antitumor
effect.(20) We also use MP as an antioxidant. However, 200 μg/ml
MP did not show cytotoxicity in 4T1 (Fig. 1). Nevertheless, thecombination treatment with MP and CPA enhanced the cytotoxi‐
city (Fig. 3D). We considered two different cases. One is the acti‐
vation of the cytochrome P450 (CYP). CYP2B6 is catalase ofCPA.(17,21) CPA is a pro-drug that is metabolized by CYP2B6 tobecome cytotoxic and effective in cancer treatment.(22) The 4-4hydroxylation product is the active metabolite of CPA that yields
an active alkylating agent, phosphoramide mustard. We analyzed
the CYP2B6 expression in 4T1 cells, however CYP2B6 expres‐sion of combination treatment with CPA and MP have nodifferent compared to control (data not shown). We cannot
conclude that CYP2B6 activity is dependent on the CYP2B6
expression. At least, the combination treatment with CPA andMP have no effect the expression of CYP2B6.
Another case is the interaction between CPA and MP. By TLC
assay, the spot of CPA with MP is different from the spot of CPA
alone (Fig. 6A). The spot of CPA was showed 300–350 pixels,
while the spot of CPA with MP was showed 250–300 pixels (Fig.
6B and C). Moreover, MP with CPA spot is also different fromMP alone spot (Fig. 6A, D, and E). The spot of 250–300 pixels
was overlap with MP and CPA. We thus concluded that CPA wasinteracted with some MP component present in 250–300 pixel.
We considered that the interaction between MP and CPA candoi: 10.3164/jcbn.20-197

A

B

C
600

D

E

Intensity (arb. units)

Intensity (arb. units)

600

CPA
CPM+MPMPCPA CPM+MP MP
Fig. 6.

CPA

CPM+MP MP

400

Distance (pixel)

Distance (pixel)

400

CPA
CPM+MPMP2000CPA CPM+MP MP

2000

TLC assay. (A) The bright field image. The extraction of CPA (B) and its line profile (C). The extraction of MP (D) and its line profile (C).

produce some cytotoxicity compounds or accelerate intracellular
CPA accumulation.
We reported the antioxidant effect of MP. MP induce cancer
specific apoptosis via accumulation of ceramide.(16) This directly
cell death was induced reduction of ROS production. On theother hands, cells were exposed 200 μg/ml MP in this study. This
concentration did not show the cytotoxicity, however the cytotox‐
icity of CPA was enhanced. We demonstrated that antioxidant
can enhance the cytotoxicity of anticancer drugs indirectly. We
have already reported that antioxidants treatments upregulate

ABC transporter expression.(23) Since ABC transporter is efflux
transporter of anticancer drugs,(24) these upregulation of ABC
transporter attenuates the cytotoxicity of anticancer drugs. In
conclusion, the cytotoxicity of cyclophosphamide treatment isenhanced by the combination treatment with MP.
Conflict of Interest
No potential conflicts of interest were disclosed.

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doi: 10.3164/jcbn.20-197