Chalcone (1,3-diphenyl-2-propen-1-one) induces apoptosis of
HeLa CD4+ cells through caspase signaling pathways
Rumiko SAITO 1), Kahoko HASHIMOTO 1), and Naoko MIYAMO-KUROSAKI 1) *Abstract
Chalcone (1,3-diphenyl-2-propen-1-one), a precursor of all flavonoids, is distributed widely among
higher plants, and chalcone derivatives have been reported to exhibit antitumor activity in vitro as well as
in vivo. However, few reports have detailed the mechanism through which chalcone induces apoptosis.
Thus, this study was undertaken to investigate the apoptotic effect of chalcone on cervical cancer cells in
vitro. The effect of chalcone on HeLa CD4+ cell viability was evaluated with the CCK-8 assay, which
revealed that chalcone can reduce the viability of HeLa CD4+ cells. Morphological and biochemical assays,
including phase contrast and PI staining fluorescence microscopy, were used to examine apoptosis
induction. Both fluorescence and phase contrast microscopy showed that 24 h treatments with chalcone
induced typical apoptotic features in HeLa CD4+ cells, including chromatin condensation, DNA
fragmentation, and apoptotic body formation. Annexin V/PI assays were performed using flow cytometry
to quantitatively test the effect of chalcone on apoptosis, revealing that chalcone induced apoptosis of
HeLa CD4+cells. Finally, activity of caspase-8, -9, and -3 was induced by chalcone treatment, indicatingthat chalcone induces apoptosis of HeLa CD4+ cells via the caspase signaling pathway.

Keywords：Chalcone (1,3-diphenyl-2-propen-1-one), Apoptosis, HeLa CD4+ cells
Received September 19, 2021; accepted October 11, 2021

1) Department of Life and Environmental Sciences, Graduate School of Engineering, Chiba Institute ofTechnology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan*Corresponding author: Naoko Miyano-Kurosaki, Department of Life and Environmental Sciences, Graduate
School of Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan
E-mail: kurosaki.naoko@p.chibakoudai.jp44

1 Introduction
In 2018, there were an estimated 569,800 new

mainly found in their hydroxyl form, have beencases of and 311,400 deaths caused by cervicalchalcone, has been reported to both inhibit tumor

cancer worldwide1,2) . Cervical cancer is both thepromotion and metastasis as well as induce apoptosis

third most frequently diagnosed cancer and the thirdin several cancer cell lines18,19) . Similarly,

leading cause of cancer-related deaths following

isoliquiritigenin and licochalcone A have beenbreast and lung cancers2) . Treatments for cervicaldemonstrated to arrest the cell cycle and induce

cancer include surgery, radiation therapy, andapoptosis in various cancer cells20,21) . The synthetic

chemotherapy (i.e., anticancer drug treatment)

3,4)

reported previously14,17) . Xanthoangelol, a natural.

chalcone

However, many of those treatments have serious side

naphthylchalcone((2E)-1-(3,4,5-

trimethoxy-phenyl)-3-(1-naphthyl)-2-propene-1-

5)effects and negatively affect quality of life (QOL) .

one) has been found to induce apoptosis and block

Many plants have antitumor pharmacologicalcell cycle progression by triggering both intrinsic

effects, and about two thirds of current anticancer

andagents,malignancy cells22) . Similarly, synthetic chalcone

including Topotecan,

Etoposide,

andextrinsic

pathwaysagainsthematologic

Paclitaxel, are compounds originally isolated from

(2,3,4′-trimethoxy-2′-hydroxy-chalcone)

plants6) . Some flavonoid compounds found among

apoptosis in human hepatocellular carcinoma cells

various foods and herbal medicines have anti-tumor

through a caspase-dependent intrinsic pathway23) .

effects7,8) with fewer side effects9,10) . For example,

Butein has been reported to suppress cervical cancer

the naturally occurring chalcones 4-hydroxyderricin,

via the PI3K/AKT/mTOR pathway24) . In contrast,

xanthoangelol, and ashitaba chalcone isolated froman analysis of the central structure of chalcone

the roots of Angelica keiskei (Miq.) Koidz

suggests that initiation may be involved in the(Umbelliferae) showed antitumor activity11) . In

antiproliferative activity of chalcone in which

Japan, the aboveground portions of this plant are

apoptosis is induced through mitochondrial andconsumed as food12). The basic structure of ashitaba

death receptor pathways in human breast cancer

chalcone (1,3-diphenyl-2-propen-1-one) is amongcells25) . In human bladder cancer cells, chalcone has

the most important classes of flavonoids foundbeen shown to inhibit cell proliferation by inducing

throughout the whole plant kingdom13) . Collectively,

apoptosis and arresting cell cycle progression in thechalcones are an important group of polyphenols that

G2/M phase26) .

inducedcan be isolated from a wide range of foods, including

The antitumor activity of chalcone in cervical

spices, fruits, teas, vegetables, and even beer14,15) .

cancer cells is associated with the cancer cell growth

Chalcones are open-chain precursors involved inpromoter tetradecanoylphorbol-13-acetate (TPA) inflavonoid and isoflavonoid biosynthesis, and they

HeLa CD4+ cells and its inhibitory activity against

occur mainly as polyphenol compounds that change

phosphorylation12) . Chalcone has also beenfrom yellow to orange in color13) . The chemical

demonstrated to have chemopreventive effects

structure of 1,3-diaryl-2-propen-1-one consists ofthrough enhancement of the antitumor activity of

two aromatic rings linked by a 3-carbon α, β-

TRAIL27) .
However, the apoptotic pathway by whichchalcone acts in cervical cancer cell HeLa CD4+ cells
is poorly understood. Accordingly, we aimed toelucidate the apoptotic pathway in which chalcone
has antitumor activity against HeLa CD4+ cells.

unsaturated carbonyl

system16). Multiple reports

have detailed the antitumor effects of the many typesof natural and synthetic chalcones. The biologically
active properties of natural chalcones, which are45

2 Experimental
2.1. Cells and reagents. HeLa cells stably

PBS containing 2% bovine serum albumin, after

transfected with human CD4+ were generously

Salamanca) was added to the resulting cell pellet.

supplied by Prof. Naoki Yamamoto (Tokyo Medical

Following a 15 min incubation at room temperature,and Dental University). The cells were cultured at

populations of PI-positive cells were analyzed with

37°C with a 5% CO2 humidified atmosphere in

a BD FACSCalibur flow cytometer (BD Biosciences,

Dulbecco’s modified Eagle’s medium (Sigma-

Franklin Lakes, NJ). The experiment consisted of

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

three or more independent replicates.

which 300 μL of PI / RNase Solution (Immunostep,

fetal bovine serum (Cytiva, Tokyo), 100 units/mL

2.4.

Assaying

nuclearmorphological

penicillin G and 100 μg/ml streptomycin (Fujifilm

changes by propidium iodide staining. The

Wako Pure Chemical Corp, Osaka). Chalcone (1,3-nuclear morphological changes caused by apoptosis

diphenyl-2-propen-1-one, purity > 98.0%) was

were examined using propidium iodide staining 24 h

procured from Tokyo Chemical Industry Co., Ltd.

after chalcone treatment. Briefly, 104 cells per well

(Tokyo) (Fig. 1A), and dimethyl sulfoxide (DMSO)

were seeded into 96-well plates. Following a 24 h

was purchased from Fujifilm Wako Pure Chemical

incubation, the cells were treated with different

Corp (Osaka).

doses of 50 μmol/L chalcone for 24 h, while the2.2. Cell viability assays. Cell viability wasvehicle control used 0.1% DMSO instead. Aftermeasured using cell counting kit-8 (CCK-8)

treatment, cells were washed twice with 1× PBS and(Dojindo Molecular Technologies, Inc., Kumamoto)stained in darkness with 10 μg/mL propidium iodide

in accordance with the manufacturer’s instructions.

(in PBS) for 10 min at room temperature. Cells wereIn brief, in addition to negative control wells

viewed at 515 nm under a fluorescent microscope(without cells), 104 cells per well (in 100 μL of(Keyence Co., Osaka,).

medium) were seeded into 96-well plates, with six

2.5. Apoptotic cell death detection by flow

replicate wells assigned to each group. After a 24 hcytometry. An Annexin V-FITC apoptosis kit

incubation, the cells were treated with different(Medical & Biological Laboratories Co., LTD.,

doses of chalcone (0, 20, 40, 60, 80, or 100 μmol/L),

Tokyo) was used to identify phosphatidylserine

and incubated again for 24 h, with 0.1% DMSO used

externalization during the process of apoptosis,

as the vehicle. After adding 10 μL of CCK-8 solution

according to the manufacturer’s instructions. Briefly,

to each well and incubating for 3 h in a dark

the HeLa CD4+ cells (3 × 105 cells per well) wereincubator at 37°C, the 450 nm absorbance values

seeded into 6-well plates and incubated for 24 h prior(OD450 values) were measured using a microplate

to experimentation and then exposed to 50 μmol/L

reader (Bio-Rad Laboratories, Hercules, CA)chalcone or 0.1% DMSO for 24 h. The cells wereaccording to the manufacturer’s instructions.

washed twice after the incubation, with PBS, and2.3. Cell cycle analysis. HeLa CD4+ cells werethen resuspended in 42 μL of binding buffer. The

seeded into 6-well cell culture plates (106 cells per

resulting cell suspensions were incubated with 2.5well) and cultured for 24 h. Then, 50 μmol/L

μL of propidium iodide and 5 μL of annexin V-FITC

chalcone was added, and cells were harvested afterat room temperature in darkness for 15 min. Annexin

another 24 h. Cells were afterwards washed with

V-positive cell populations were directly analyzed

phosphate-buffered saline (PBS), fixed with 70%

using BD FACSCalibur. The experiment wasethanol for 30 min at 4°C, and washed again with

conducted with three or more independent replicates.
46

2.6. Caspase-8 and -9 activity assay.
Caspase-8 and -9 activity was measured using
Caspase-8 and -9 FITC staining kits, respectively(Abcam,Cambridge)

accordingtothemanufacturer’s instructions. In brief, 106 cells wereincubated in 6 cm dishes with 50 μmol/L chalcone.
Caspase activity rates were analyzed using BD
FACSCalibur. The experiment consisted of three ormore independent replicates.

2.7. Caspase-3 activity assay. Caspase-3
activity was measured with the Caspase-3 (active)
FITC staining kit (Abcam, Cambridge) according to
the manufacturer’s instructions. Briefly, 3 × 105 cells
were incubated in 6-well plates with 50 μmol/L
chalcone. Caspase activity rates were monitored
using BD FACSCalibur. The experiment consistedof three or more independent replicates.

2.8 Statistical analysis. All data were processed

Fig 1. Inhibitory effect of chalcone on HeLa
CD4+ cell growth. (A) Chemical structure of 1,3diphenyl-2-propen-1-one (chalcone); (B) Cell
viability was determined by CCK-8 assay.

using Microsoft Office Excel (Microsoft, Redmond,
WA). All results are expressed as means ± standard
deviation (SD) and each experiment was repeatedindependently at least 3 times. In addition, statisticaldifferences were analyzed by Student’s t-test. Statistical

3.2. Chalcone increased the proportion ofsignificance was indicated by P < 0.05 (both sides).

HeLa CD4+ cells in sub-G1 phase. Flowcytometry was used to quantify cells in each cell

3 Result
3.1. Cytotoxic effects of chalcone inhibited

cycle phase after the addition of chalcone. In thecontrol, the percentage of HeLa CD4+ cells in sub-HeLa CD4+ cell growth. The effect of chalcone on

G1 phase was 4.9 ± 0.5%. In contrast, the 24 h 50

HeLa CD4+ cell viability was determined by treating

μmol/L chalcone treatment significantly increased

the cells with various concentrations of chalcone (0the percentage of cells in sub-G1 phase to 17.3 ±μmol/L to 100 μmol/L) for 24 h and evaluating

1.0% (Fig. 2). The percentage of cells in the G0/G1

subsequent cell viability using the CCK-8 assay (Fig.
1B). The

additionofchalconeatphase was significantly reduced from 53.6 ± 1.0% in

variousthe control to 42.1 ± 2.3% in the 24 h 50 μmol/Lconcentrations over 24 h affected HeLa CD4+ cells,

chalcone treatment (Fig. 2). For the other cell cycle

resulting in a concentration-dependent decrease inphases, including the S phase and G2/M phase, thecell viability (Fig. 1B). After 24 h of chalcone

addition of chalcone did not significantly change intreatment, up to 50% cell death was observed at a

HeLa CD4+ cells.

dose of 51.8 ± 4.3 μmol/L, and so subsequent
experiments were performed with a chalcone
addition of 50 μmol/L.
47

Fig 3. Morphological observation by propidium
iodide (PI) staining. Morphology was observed
from PI-stained phase contrast images (left), anda fluorescence image (center) and a merge image(right) were thus obtained. (A) to (C) show
chalcone-free cells, while (D) to (F) show cells
to which 50 μmol/L chalcone was added. (A–F)
show cells 24 h after chalcone addition.
Morphological change in cells were observed by
microscopy. Scale bar, 50 μm.

Fig 2. Effect of chalcone on the cell cycle of
HeLa CD4+ cells. (A) HeLa CD4+ cells were
stained with propidium iodide (PI), the DNAcontent in the cells was measured by flow
cytometry, and the cell cycle phase of each cell
was recorded as sub-G1, G0/G1, S, or G2/M.
(B) Percentage of cells distributed among
different cell cycle phases. Data are expressed asmean ± SD values of three independent
experiments. Significant differences between
chalcone-treated cells and the control (vehicle)
group are labeled (** P < 0.01 or *** P < 0.001).

3.3.

Chalcone

inducedmorphologicalchanges characteristic of apoptosis in HeLa
CD4+ cells. PI staining was used to examine

Fig 4. Induction of HeLa CD4+ cell apoptosis bychalcone. (A) Apoptosis rates were determinedby flow cytometry analysis using annexin VFITC/PI double labeling. HeLa CD4+ cells weretreated for 24 h in the absence or presence of 50
μmol/L chalcone. (B) Histogram of quantitative
analysis of dead (UL), late apoptotic (UR),viable (LL), and early apoptotic (LR) control
HeLa CD4+ cells or HeLa CD4+ cells treatedwith 50 μmol/L chalcone for 24 h. Values are
expressed as mean ± SD values of three
replicates. * P < 0.05 or ***P < 0.001 comparedwith the untreated control (vehicle) group.

chalcone-induced apoptosis. HeLa CD4+ cells weretreated with chalcone for 24 h (Fig. 3). Cellshrinkage, apoptotic bodies, nuclear condensation,and nuclear fragmentation, each of which are typicalmorphological features of apoptosis, were observed.
This result was consistent with the results of a study
examining the effect of chalcone addition on the cell
cycle, which showed an increased percentage of cellswith fragmentation of the nuclei in the sub-G1 phase.
48

3.4. Chalcone induced apoptosis of HeLa

apoptotic or necrotic cells was 3.9 ± 1.3% underCD4+ cells. Using flow cytometry and an annexin

control conditions, the apoptosis rate induced by the

V-FITC / PI double labeling assay, we quantified the

24 h incubation with chalcone was significantly

percentage of apoptotic cells 24 h after the addition

increased to more than 11 times higher, at 46.0 ±of chalcone to HeLa CD4+ cells. Based on the

8.7% (Fig. 4B).

resulting flow cytometry data, each stained cell was

3.5. Chalcone induced caspase-dependentclassified into one of four groups: (1) viable (LL),cell death in HeLa CD4+ cells. We also examined(2) early apoptosis (LR), (3) late apoptotic orwhether caspases are major molecular regulators of

necrotic (UR), and (4) dead cells (UL). The

cell death. Data are presented as a percentage ofpercentage of late apoptotic cells and early apoptotic

chalcone-free controls (Fig. 5). After chalcone

cells significantly increased after the 24 h incubation

treatment, the activity levels of caspase-8, -9, and -3

with chalcone (Fig. 4A). While the percentage of lateincreased from 9.9 ± 2.0% to 35.5 ± 3.4%, from 9.1

Fig 5. Caspase-8, -9, and -3 activity levels and effects of caspase inhibition on chalcone-treated HeLa CD4+
cells. Caspase-8 (A), -9 (B), and -3 (C) activity was measured by flow cytometry in HeLa CD4+ cells after
a 24 h chalcone treatment. The apoptotic cell populations for active caspase-8, caspase-9, and caspase-3
were quantitatively analyzed (left) and graphed (right). Data are represented as mean ± SD values of threeexperiments. *** P < 0.001 compared with the untreated control (vehicle) group.
49

± 3.9% to 41.1 ± 3.4%, and from 6.7 ± 1.6% to 32.2

cells, and the viability of HeLa CD4+ cells was 50%± 2.7%, respectively.

after the 24 h approximately 50 μmol/L chalcone
treatment (Fig. 1). Subsequently, cell cycle analysis

4 Discussion

was performed in order to examine the effect ofAmong women aged 15 to 44, cervical cancer ischalcone on the cell cycle of HeLa CD4+ cells. No

the second most common cancer and cancer-related

significant

cause of mortality1) . Cervical cancer onset andpercentages of cells in the S phase and the G2/M

treatment side effects can significantly impact

phase between chalcone-treated cells and control

patient QOL28) . So, it is important to develop new

cells. However, significantly more chalcone-drugs with few side effects. Apoptosis plays an

supplemented cells were in sub-G1 phase comparedimportant role in various diseases, including as a

to control cells (Fig. 2). Furthermore, in order totarget and side effect of many cancer treatments, andconfirm whether the effect of chalcone on sub-G1

presents an approach to modulating related apoptotic

phase cells was related to apoptosis, HeLa CD4+

pathways in cancer drug discovery research29,30) .

cells were subjected to PI staining 24 h after the

Drugs that restore or modulate normal apoptotic

addition of chalcone, serving as a morphological cell

pathways allow for the effective treatment of cancers

death assay. Cells in the control group exhibitedthat are dependent on abnormalities in apoptotic

normal morphology (Fig. 3A), but after addition ofpathways31) . Accordingly, as a drug identification

chalcone, HeLa CD4+ cells exhibited contraction

strategy, it is useful to find anticancer drugs thatand apoptotic bodies in phase contrast images (Fig.

promoteapoptosis32)

difference

wasobservedinthe. Ashitaba is a common

3D). In the early course of apoptosis, contraction andvegetable and medicinal plant containing many

concentration of cells is observable with an optical

phytochemicals, including the naturally occurring

microscope34). Cell contraction reduces cell size andchalcones 4-hydroxydericin, xanthoangelol, andincreases the density of the cytoplasm, causing theashitaba chalcone, each of which can be isolatedorganelles to become more densely packed; pyknosis

from the plant’s roots11). The anticancer activity ofoccurs as a result of chromatin condensation, which

synthetic chalcone utilizing the compound’s basic

is one of the morphological features of apoptosis35).

structure has also been widely studied33) . However,Subsequently, PI staining was conducted to assess

efforts to explore the mechanisms by which the basic

changes in the nucleus. The nuclei of cells

structure of chalcone is cytotoxic to cervical cancer

undergoing apoptosis were examined for nuclear

cells, and more generally, the anticancer activity ofcondensation by fluorescence microscopy (Fig. 3E).

chalcone against cancer cell lines, have been

These results indicated that chalcone prominently

insufficiently examined. Therefore, we aimed toinduces apoptosis in HeLa CD4+ cells. The

elucidate the mechanism of apoptosis induced byfluorescence microscopy analysis of PI stained cells

chalcone in HeLa CD4+ cells, a widely researchedindicated that chalcone is an inducer of cell apoptosis,

cervical cancer cell line, and this study was more

and the flow cytometric analysis of cells stained withbroadly conducted to provide biochemical evidence

annexin V-FITC and PI revealed the relevance offor the apoptotic activity of chalcone.

apoptosis induction. On the surface of apoptotic cells,

First, the cytotoxicity of chalcone to HeLa CD4+
cells

was

examined.

Chalconethe normal inward phosphatidylserine of the cell’s

exhibited

lipid bilayer migrates and is expressed in the outerconcentration-dependent cytotoxicity to HeLa CD4+

layer of the plasma membrane36). Annexin is a50

recombinant phosphatidylserine-binding protein that

The design of anticancer agents that target the

specificallywithchalcone-induced apoptotic pathway may provide anphosphatidylserine residues and is therefore used toeffective treatment with few side effects. Designingandstronglyinteracts

37,38)

detect apoptosis. In addition, the membrane-

a drug by targeting the chalcone-induced apoptotic

impermeable nucleic acid dye propidium iodide (PI)pathway as a cancer therapeutic drug might enablepasses through the cell membrane of apoptotic cells

the production of a drug that is more effective and34)

that are collapsing . Therefore, double positive

has fewer side effects. Natural products are gaining

results for Annxin V and PI suggest apoptosis. Flow

increasing attention due to their potential anti-cancer

cytometrytheactivity and low intrinsic toxicity. In addition toaccumulation of apoptotic cells in the upper rightchalcone, attention has been paid to the anticancer(UR) quadrant after chalcone treatment (Fig. 4).

activity of flavonoids. Epigallocatechin gallate

Furthermore, the annexin V-FITC and PI staining(EGCG), which is the main component of green tea

results were consistent with each other, confirming

polyphenol, has been reported to have anticancer

that chalcone induced apoptosis in HeLa CD4+ cells.

activity against breast cancer40). Zan et al. reported

Apoptosis is tightly controlled by anti- and pro-that EGCG induced apoptosis in breast cancer MCF-

apoptotic proteins and is promoted by various

7 cells through activation of caspase-9, caspase-3,

pathways28). Apoptotic cells exhibit biochemicaland poly (ADP-ribose) polymerase-1 cleavage41).

modifications such as protein cleavage, protein

Also Kwak et al. found that EGCG inducescross-linking, DNA degradation, and phagocytosis

apoptosis in human cholangiocarcinoma HuCC-T1

recognition. The proteolytic cascade caspase cancells through an increase in the apoptosis-promoting

activate other caspases, leading to cell death byproteins Bax, activation of caspase-9 and caspase-3,

amplifying the signal transduction pathway ofand release of cytochrome c42).

ofapoptotic

cells

revealed

apoptosis39). Therefore, to identify the apoptotic

In the future, comprehensive elucidation of thepathway induced by chalcone treatment, caspase

effects of chalcones on cancer cells at the DNA andexpression was examined. The protein expression

mRNA levels should be a major focus of drug

levels of caspase-8, -9, and -3 by HeLa CD4+ cells

research and development.

increased after chalcone treatment (Fig. 5). Induction

Acknowledgments. We would like to thank Ms.

of apoptosis in HeLa CD4+ cells by chalcone
indicates that caspase-3 was activated by an

S. Mitsutake for helping us with this experiment.

upstream cascade including caspase-8 and caspase-9

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