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MATERIAL TECHNOLOGY
材料技術

March-April
2023
Vol. 41, No. 2
ISSN 0289-7709
CODEN: MTECFQ

材料技術研究協会
Japan Research Institute ofMaterial Technology
https://www.jrimt.jp

MATERIALTECHNOLOGY
材料技術

VOL. 41, NO. 2
MARCH-APRILCONTENTS＜ARTICLES＞
ANTITUMOR EFFECT OF VIOLACEIN ON COLORECTAL CANCER CELLSTokimasa MATSUI, Atsuko TSUKIMOTO, Kahoko HASHIMOTO, Yasuyuki
TAKIGUCHI, Naoko MIYANO-KUROSAKI·················································9<EDITORIAL NOTE>
編集後記···························································································17

Antitumor effect of violacein on colorectal cancer cells
Tokimasa MATSUI1, *, Atsuko TSUKIMOTO2, *, Kahoko HASHIMOTO1, Yasuyuki TAKIGUCHI1
and Naoko MIYANO-KUROSAKI1, **Abstract
Violacein is a blue-purple pigment extracted from soil bacteria and has been reported to have anticancer
activity. In this study, we assessed the anticancer activity of violacein and confirmed the cytotoxicity andinduction of apoptosis in colon cancer cell lines. It was confirmed that violacein-induced apoptosis
follows two pathways. In the first pathway, activation of caspase-8 (an inducible caspase) activates
caspase-3 (an executive caspase), which induces apoptosis in cancer cells. In the second pathway,
cytochrome c is released from mitochondria and activates caspase-9 (an inducible caspase), which in turnactivates caspase-3 (an executive caspase), which induces apoptosis in cancer cells. These findings
suggest that violacein might be useful as a therapeutic agent for colorectal cancer in the future.

Keywords：violacein, colon cancer cell, apoptosis
Received December 12, 2022; accepted June 1, 2023

1) Department of Life Sciences, Graduate School of Advanced Engineering, Chiba Institute of Technology, 2-171 Tsudanuma, Narashino, Chiba 275-0016, Japan
2) Department of Medical Technology, Faculty of Medical and Health Sciences, Tsukuba International University,
6-20-1 Manabe, Tsukuba, Ibaraki 300-0051, Japan.

*Contributed equally.
**Corresponding author: Naoko Miyano-Kurosaki, Department of Life Sciences, Graduate School of Advanced
Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan
E-mail: kurosaki.naoko@p.chibakoudai.jp

9

1. Introduction
Cancer is the leading cause of death in Japan

were seeded in 96-well plates at a density of 5 × 104
cells/well. Subsequently, cells were treated with

(27.6 %)1); in particular, colorectal cancer (CRC) has

various doses of violacein (0, 5, 10, and 20 μmol/L).

high morbidity and a high mortality rate2-3). Hence,

After 24 h of incubation, cells were incubated withthere is an urgent need to identify novel therapeutic

10 µL CCK-8 reagent at 37°C for 2 h. Absorbancedrug candidates for this disease. Therefore, we

was detected at 450 nm using the iMark™

assessed the antitumor activity of violacein, which

Microplate Reader (Bio-Rad Laboratories, Hercules,

has various physiological activities such asCA).

antibacterial, antioxidant, and anticancer effects4-7).

2.3 Apoptotic analysis. COLO205 cells were

Violacein is a natural bluish-purple pigment

seeded in 48-well plates at a density of 105 cells/well,compound (3E)-3-[5-(5-hydroxy-1H-indol-3-yl)-2oxo-1,2-dihydro-3H-pyrrol-3-ylidene]-1,3-dihydro-

followed by the addition of 1 % DMSO (control) or

2H-indol-2-one with a molecular weight of 343.348).

10 µmol/L violacein to the cells. After 24 h,Natural pigments such as violacein lack vividness

Annexin-V/PI

compared with synthetic pigments and have theLaboratories, Hercules, CA) was conducted.

drawback of low color stability (e.g., fading in

Fluorescence intensity was measured using the BD

sunlight), but they have physiological activities such

FACSCalibur Flow Cytometer (Becton Dickinson,

as antibacterial and antioxidant properties and haveFranklin Lakes, NJ). For each measurement, at least

attracted attention as functional pigments.

10,000 cells were counted.

Apoptosis

Assay(Bio-RadIn this study, we assessed the antitumor effect of

2.4 Loss of MMP. COLO205 cells were seeded inviolacein on CRC cells and determined its

48-well plates at a density of 105 cells/well. Then,mechanism of action.

1 % DMSO (control) or 10 µmol/L violacein wasadded to the cells. After 24 h, the mitochondrial

2. Experimental
2.1 Cell culture. This study was performed using

membrane potential (MMP) was measured byadding JC-1 dye (Cell Technology, Davis, CA) to thethe COLO205 human CRC cell line9). COLO205

cells and measuring fluorescence using the BD

cells were cultured in RPMI 1640 Medium (Sigma-

FACSCalibur Flow Cytometer.

Aldrich, St. Louis, MO) supplemented with 10 %(Cytiva,

2.5 Release of cytochrome c. COLO205 cells

Marlborough, MA), 100 units/mL penicillin G, andwere seeded in 48-well plates at a density of 105

100 μg/mL streptomycin sulphate (Fujifilm Wako

cells/well. Then, 1 % DMSO or 10 µmol/L violacein

Pure Chemical Corporation, Osaka). The cells were

was added, and cytochrome c was measured after 24

cultured at 37°C in an incubator with 5 % CO2.

h, using anti-cytochrome c antibody (Abcam,

Violacein was dissolved in dimethyl sulfoxide

Cambridge, MA) according to the manufacturer’s(DMSO; Fujifilm Wako Pure Chemical Corporation)

instructions. Fluorescence was measured using theand the final concentration of DMSO in the controls

BD FACSCalibur Flow Cytometer.

heat-inactivatedfetalbovineserum

was 1 %.

2.6 Detection of activated caspase. COLO205

2.2 Cell proliferation assay. Cell proliferation

cells were seeded in 48-well plates at a density of 105

was examined with Cell Counting Kit-8 (CCK-8) Kit

cells/well. Then, 1 % DMSO or 10 µmol/L violacein(Dojindo Laboratories, Kumamoto). Briefly, cells

was added to the cells. After 24 h, the activation of
10

caspase-3, caspase-8, and caspase-9 was measured
with the Cleaved Caspase-3 FITC Staining Kit,
Caspase-8 (active) FITC Staining Kit, and Caspase9 (active) Red Staining Kit (all from Abcam),
respectively, according to the manufacturer’s
instructions. Fluorescence was measured using the
BD FACSCalibur Flow Cytometer.
2.7 Autophagy detection with DALGreen
staining. Cells were plated in 24-well plates andcultured at 37°C with 1 mL of 0.1 μmol/L
DALGreen (Dojindo Laboratories) for 30 min. Afterthe cells were washed twice with culture medium,
they were incubated with or without 10 μmol/L
violacein for 48 h. Then, cells were washed twicewith PBS(-), and fluorescence was measured using
the BD FACSCalibur Flow Cytometer. For all
samples, DALGreen fluorescence was collected
through the FL1 channel and 10,000 events wereacquired in at least three separate experiments.

3. Results and Discussion
3.1 Cytotoxicity test. The CCK-8 assay was
performed to confirm that violacein is cytotoxic to
COLO205 cells. As shown in Figure 1, violacein

Fig. 2. Annexin V/PI assay of COLO205 cells at 24h after the addition of violacein (10 μmol/L).
Representative scatter plots of PI (y-axis) vs.
Annexin V/FITC (x-axis). Dot plot of detected
fluorescence control (A), DMSO (B), violacein(C). UL is cells stained only with PI, UR is necrotic
cells or late apoptotic cells stained with bothAnnexin-V and PI, LL is living cells not stainedwith either Annexin-V and PI, and LR is early
apoptotic cells stained only with Annexin V.
Analysis of the detected fluorescence ratio andgraphing (D). The experiments were performed
three times each, and the data are presented as themean ± standard deviation (SD). * P < 0.05, ** P <0.01 and *** P < 0.001 versus control.

Fig. 1. The viability of COLO205 colon cancer
cells was tested by the CCK-8 assay. COLO205
cells were seeded at a density of 5 × 104 cells/welland treated with 5 to 20 μmol/L violacein, and theabsorbance was measured after 24 h. The
experiments were performed three times each, andthe data are presented as the mean ± standard
deviation (SD).

11

Fig. 3. Analysis of the mitochondrial membrane depolarization by JC-1 dye of COLO205 cells treated with andwithout 10 μmol/ L violacein for 24 h. Representative dot plots of JC-1 dye of COLO205 cells are shown. The
lower panel shows COLO205 cells exhibiting green fluorescing monomers, representing polarized cells; and theupper panel shows cells exhibiting red fluorescence aggregates, representing depolarized cells (A). Percent
decrease in normal cells and increase in apoptotic cells based on the dot plot of JC-1 dye (B and C). The
experiments were performed three times each, and the data are presented as the mean ± standard deviation (SD).
*** P < 0.001 versus control.

decreased the viability of cells in a concentration-

two fluorescence signals were detected (Fig. 2A-D).

dependent manner, with 50 % cell death occurring at

The results showed that the population of dead cells

12.5 µmol/L. Therefore, subsequent experiments

increased about 3.7 fold in cells treated with

were performed at a violacein concentration of 10

violacein compared with the control, showing that

µmol/L. Cytotoxicity did not change over time.

violacein is cytotoxic. In addition, the proportion of

3.2 Analysis of apoptosis by fluorescence-

early apoptotic cells increased about 2.7 fold,activated cell sorting. In cells in which early

showing that violacein induces early apoptosis.

apoptosis has been induced, the nuclear envelope

3.3 Loss of MMP and release of cytochrome c.

remains normal, the asymmetry of the cell

In cells in which early apoptosis has been induced,

membrane is disrupted, and phosphatidylserine is

the intracellular MMP disappears and cytochrome c,

exposed to the outside of the cell membrane. PI emits

an apoptosis-inducing factor, is released into thered fluorescence by binding to Annexin V and thecell17 ） . Because the JC-1 dye has affinity for the

double helix of DNA in the nucleus10-16), and these

MMP, it emits red fluorescence when the MMP is12

Fig. 4. Detection of cytochrome c in COLO205 cells by flow cytometry at 24 h after the addition of violacein(10 μmol/L). Graph showing the fluorescence intensity of cytochrome c FITC superimposed for the control(dotted line), DMSO (gray), violacein (solid line) (A). Graph showing the ratio of fluorescence intensity ofcytochrome c FITC (B). The experiments were performed three times each, and the data are presented as themean ± standard deviation (SD). ** P < 0.01 versus control.

Fig. 5. Detection of caspase-8, caspase-9, and caspase-3 in COLO205 cells by flow cytometry at 24 h after the
addition of violacein (10 μmol/L). Graphs showing the fluorescence intensity of caspase-8 FITC for the control(dotted line), DMSO (gray), and violacein (solid line) (A) and the ratio of the fluorescence intensity of caspase8 FITC (B). Graphs showing the fluorescence intensity of caspase-9 Red-LEHD-FMK for the control (dottedline), DMSO (gray) and violacein (solid line) (C) and the ratio of fluorescence intensity of caspase-9 RedLEHD-FMK (D). Graphs showing the detected fluorescence intensity of cleaved caspase-3 FITC for the control(dotted line), DMSO (gray), and violacein (solid line) (E), and the ratio of fluorescence intensity of cleaved
caspase-3 FITC (F). The experiments were performed three times each, and the data are presented as the mean
± standard deviation (SD). ** P < 0.01 and *** P < 0.001 versus control.

normal and emits green fluorescence when the MMP

an antibody that specifically binds to cytochrome cdecreases17-21). The results showed that the MMP

and emits green fluorescence was used to detect thewas reduced to about 1/18 that of the control in cells

fluorescence22-27). The results showed that cells

treated with violacein (Fig. 3A-C).

supplemented with violacein released about 3.4

Next, using the BD FACSCalibur Flow Cytometer,times more cytochrome c compared with control
13

Fig. 6. Effects of violacein on autophagy. Flow cytometry analysis of autophagosome formation (autolysosome)in COLO205 cells treated with violacein for 48 h. Graph showing the fluorescence intensity of DALGreenDMSO (solid line) and violacein (gray) (A). Graph shows the ratio of the fluorescence intensity of DALGreen(B). The experiments were performed three times each, and the data are presented as the mean ± standard
deviation (SD). ** P < 0.01 versus DMSO.

cells (Fig. 4A, B). These data suggest that the MMP

condensation in Huh7 and Hep3B hepatocellular

disappeared due to the destruction of the intracellular

carcinoma cells, alters MMP, and increases reactive

mitochondrial

andoxygen species (ROS), leading to activation of thecytochrome c was released from the mitochondria

caspase cascade and induction of apoptosis. In

into the cytoplasm.

addition, Queiroz et al. 35) reported that violacein

membranebyviolacein,

3.5 Detection of activated caspase. To confirm

induces cell death in TF1 leukemia progenitor cells

the route by which violacein induces apoptosis, theby inhibiting calpain and DAPK1 and activating

activity of caspase was evaluated using an antibody

PKA, AKT, and PDK through endoplasmic

that specifically binds to cleaved caspase-3, caspase-reticulum stress and Golgi apparatus collapse. These8, and caspase-928-34). The results showed that cells

findings suggest that violacein induces apoptotic cell

supplemented with violacein had about a 6-folddeath via endoplasmic reticulum stress and Golgi

higher caspase-8 activity compared with control

apparatus collapse due to increased nuclear

cells (Fig. 5A, B). Cells supplemented with violacein

aggregation and ROS.

had about an 8-fold higher caspase-9 activity

3.6 Effects of violacein on autophagy. Our

compared with control cells (Fig. 5C, D). Cells

previous study found that violacein induced cell death

supplemented with violacein had about a 6-fold

due to necrosis as well as apoptosis5). In addition, it ishigher caspase-3 activity compared with control

known that autophagy is a mechanism that cancells (Fig. 5E, F). Together, these results showed that

regulate apoptosis positively or negatively36-39).

violacein activates inducible caspase-8, which

Therefore, we studied the effects of violacein on

stimulates

inducing

autophagy. Cells were treated with 10 µmol/L

apoptosis and the release of cytochrome c from theviolacein for 48 h to label autophagosomes.

mitochondria. Thus, inducible caspase-9 andFluorescence-activatedexecutive caspase-3 are activated to induce apoptosis

demonstrated

by two routes that cause cancer cells to undergo

significantly increased autophagy in COLO205 cells

apoptosis.

(Fig. 6A, B).

caspase-3

activity,thus

Kim et al.7) reported that violacein induces nuclear
14

that

cell

FACS

analysistreatment

withviolacein4. Conclusion

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In this study, we investigated whether violacein

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CRC cells. The CCK-8 assay showed that violacein

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Moore, Cancer Res., 1978, 38, 1345-1355.

caspase-3, which was activated by caspase-8 or

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induced a significant increase in apoptosis andRes., 1996, 22, 4592-4593.

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16

MATERIAL TECHNOLOGY 材料技術
昭和62年8月5日第三種郵便物許可

令和5年4月25日発行（隔月25日発行）

第41巻

第２号

定価2,100円