Research Paper

Shikonin Induces Oxidative Damage and Promotes Cell

Senescence in Lung Cancer Cells through p53/p21WAF
Signaling Pathway
LIWEN RONG, YUXIN HE, MENGZHEN YUAN, L. ZHENG, TINGTING ZHAN, HUA LING AND JUN ZHANG*
Department of Oncology, The Third People's Hospital of Chengdu, Chengdu, Sichuan Province 610031, China

Rong et al.: Effect of Shikonin on the Oxidative Damage of Lung Cancer Cells

To investigate the effect of shikonin on the oxidative damage of lung cancer cells and promotes cell
senescence through p53/p21Waf signaling pathway. The lung adenocarcinoma A549 cells were cultured
and made into cell suspension. The lung adenocarcinoma A549 cells were cultured for 12 h, 24 h and 48
h respectively with different concentrations of shikonin (0 μm, 2 μm and 4 μm) and the activity of lung
adenocarcinoma A549 cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
method. The cell cycle of each group was measured by flow cytometry after 48 h of action of shikonin. The
aging of each group of cells was determined by beta-galactosidase staining after 48 h of action of shikonin.
The reactive oxygen species content in each group was determined by flow cytometry 48 h after the action
of shikonin. The expression levels of cyclin D1, demethylase KDM2B, deoxyribonucleic acid damage
marker protein p-H2AX, p53 and p21WAF were measured by Western blot. With the increase of shikonin
concentration, the cell activity and the expression levels of cyclin D1 and KDM2B were significantly
decreased, the proportion of resting phase/intermediate phase cells and senescent cells were significantly
increased, the level of reactive oxygen species and the expression levels of p-H2AX, p53 and p21WAF were
significantly increased (p<0.05). Shikonin can inhibit cell activity, induce oxidative damage and promote
cell senescence, which may be achieved by activating p53/p21WAF signaling pathway.
Key words: Shikonin, p53/p21waf signaling pathway, lung cancer, oxidative damage, cell aging

Lung cancer, also known as primary bronchogenic cancer. Shikonin is an important active ingredient
carcinoma, is one of the most common malignant of Lithospermum. Some studies have found that the
tumors in the lungs. According to statistics, more than shikonin could promote programmed cell necrosis
10 million patients die from lung cancer each year and in glioma cells and breast cancer, and play a role of
more than 1.2 million new cases have emerged every clearing heat, detoxifying and anti-inflammatory, etc.
year. Whereas the 5 y survival rate of lung cancer In addition, the shikonin may also induce oxidative
patients is only about 16 %[1]. With the development stress[4,5]. However, its mechanism and roles in lung
of new effective anti-cancer drugs and the increase cancer are not clear. The purpose of this study is to
of new treatment regimens, the curative effect of investigate the effect of shikonin on oxidative damage
chemotherapy has been greatly improved. At present, and cell senescence of lung cancer cells and to discuss
the comprehensive treatment of non-small cell lung its mechanism.
cancer is mainly combined with radiotherapy and
chemotherapy. However, the recurrence rate is high, MATERIALS AND METHODS
the adverse reactions caused by chemotherapy are quite
Experimental cells:
a lot and the prognosis is poor[2]. Traditional Chinese
medicine is the traditional treasure of China. A variety of Cell line A549 of human non-small cell lung cancer
active ingredients of traditional Chinese medicine have
a certain anti-tumor effect. Currently, the traditional This is an open access article distributed under the terms of the Creative
Chinese medicine has become an important means Commons Attribution-NonCommercial-ShareAlike 3.0 License, which
allows others to remix, tweak, and build upon the work non-commercially,
to treat advanced lung cancer. In recent years, many as long as the author is credited and the new creations are licensed under
studies have confirmed the advantages and status[3] of the identical terms

traditional Chinese medicine in the treatment of lung Accepted 06 July 2022

Revised 14 December 2021
Received 05 June 2021
*Address for correspondence
Indian J Pharm Sci 2022;84(4):832-837
E-mail: hsrlw@163.com
832 Indian Journal of Pharmaceutical Sciences July-August 2022
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(Wuhan Procell Life Science & Technology Co., Ltd.) measured by flow cytometry in each group 48 h after the
action of shikonin. The cell suspension was taken and
Major instruments and reagents: cultured for 48 h with different concentrations (0 µM, 2
Low-temperature high-speed centrifuge (Changsha µM and 4 µM) of shikonin, washed by phosphate buffer
Dongwang Experimental Instruments Co., Ltd.); and centrifuged. The supernatant was removed and 1
ultra-low temperature refrigerator (Zhejiang Jisheng ml of phosphate buffer was added and suspended again.
Cryogenic Equipment Co., Ltd., model: DW-86W300); Then 600 μl Propidium Iodide (PI) was added in, gently
paraffin slicer (Beijing Shengkexinde Technology Co., vortexing and blending. The cells were incubated at 37°
Ltd., model: RM2255); electron microscope (Beijing away from light for 0.5 h and fully mixing for computer
Jingkerida Technology Co., Ltd., model: EM208s); testing.
electronic balance (Changsha Dongwang Experimental Determination of cellular senescence: The cell
Instruments Co., Ltd., model: TG20KR-D); fetal bovine senescence of each group was determined by beta (β)-
serum (Shanghai Jianglin Biotechnology Co., Ltd.); galactosidase staining 48 h after the action of shikonin.
anhydrous ethanol (Dongguan Wanxin Fine Chemical The cell suspension was taken and A549 cells were
Co., Ltd.); reverse transcriptome kit (Shanghai Yucan cultured for 48 h with different concentrations (0 µM,
Biotechnology Co., Ltd.) and shikonin (Chengdu 2 µM and 4 µM) of shikonin. The fixed solution was
Herbpurify Co., Ltd., purity over 98 %). added at room temperature for 10 min. It was washed by
phosphate buffer solution and stained by X-gal staining
Cell culture:
solution. β-galactosidase-positive cells were observed
The A549 cells were cultured in Dulbecco's Modified by microscope and the percentage of positive cells
Eagle Medium (DMEM). The cells in good growth was calculated, namely, the proportion of senescent
condition and in logarithmic phase were made into cells (positive cell ratio=positive cell number/total cell
cell suspension. The cell suspension was taken and number×100 %).
centrifuged repeatedly for 3 times, cultured in an Determination of Reactive Oxygen Species (ROS):
incubator containing 10 % fetal bovine serum, 5 % The content of ROS in each group was determined by
carbon dioxide at 37°. The culture medium was changed flow cytometry 48 h after the action of shikonin. The
daily. The trypsin was added for digestion when the cells cell suspension was taken and A549 cells were cultured
grow to about 80 %. The culture was terminated when for 48 h with different concentrations (0 µM, 2 µM and
the cells became round, then gently blown until the cells 4 µM) of shikonin. The trypsin was added for digestion.
fell off completely. The cell suspension was repacked It was washed by phosphate buffer solution. The cells
into a new culture flask and cultured in an incubator were re-suspended by adding 1 ml of phosphate buffer
containing 10 % fetal bovine serum, 5 % carbon dioxide and then adding 1 μl of stain. It was incubated at 37°
at 37°. The cells were counted and inoculated into for 0.5 h and centrifuged. The supernatant was removed
cryopreservation tube with the concentration of 10 000 and 1 ml of the phosphate buffer solution was added for
cells/ml. It was slowly frozen at 4° first, then placed in computer testing.
an ultra-low temperature refrigerator at -80° for 1 h and
finally put into the liquid nitrogen standby for testing. The expression levels of cyclin D1, demethylase
KDM2B and Deoxyribonucleic Acid (DNA) injury
Observation indicators: marker protein p-H2AX, p53 and p21waf in each
group were determined by Western blotting. The cell
Determination of cellular activity:
suspension was taken and cultured with different
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
concentrations (0 µM, 2 µM and 4 µM) of shikonin
bromide (MTT) method was used for the detection of
for 48 h and then the cell lysis buffer was added for
the change of A549 cell activity. Cell suspension was
full pyrolysis. The supernatant was taken after being
taken and the A549 cells were cultured with different
centrifuged at 4°, namely, the total cell protein. The
concentrations (0 µM, 2 µM and 4 µM) of shikonin for
sample protein was taken to calculate the sample
12 h, 24 h and 48 h. Then it was cultured for 2 h at 37°
volume. It was heated for protein denaturation. The
after adding 5 mg/ml of MTT solution. The absorbance
separation gel and spacer gel were put in to go through
value at 470 nm was determined by Enzyme-Linked
loading, electrophoresis, trarsmembrane and sealing.
Immunosorbent Assay (ELISA).
Primary antibodies were added and incubated at 4°.
Cell cycle measurement: The change of cell cycle was The horseradish peroxide-cohered goat polyclonal

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secondary antibodies to rabbit Immunoglobulin G significant.
(IgG) was added and incubated at room temperature.
The chemiluminescence solution was added. It was RESULTS AND DISCUSSION
placed in a luminous machine for exposure and storage The cell activity decreased significantly (p<0.05) in the
of strips. The image grayscale analysis was carried out 2 µM shikonin group compared with the 0 µM shikonin
using Quantity One. group and the cell activity decreased significantly
Statistical methods: (p<0.05) in the 4 µM shikonin groups compared with
the 2 µM shikonin group. Cell activity in each group
The comparison of measurement data was accordance decreased significantly (p<0.05) with the increase of
with normal distribution, including cell activity, cell shikonin concentration and extension of time as shown
proportion of Resting phase/Intermediate phase (G0/ in Table 1 and fig. 1.
G1), percentage of senescent cells, ROS content, and
The percentage of cells in G0/G1 was significantly
expression levels of cyclin D1, KDM2B, p-H2AX, p53
increased (p<0.05) in the 2 µM shikonin group 48 h
and p21waf in each group of this study. The comparison
after the action of shikonin compared with the 0 µM
among multiple groups was based on univariate multi-
shikonin group and the percentage of cells in G0/G1 was
sample mean. The independent sample t test was used
significantly increased (p<0.05) in the 4 µM shikonin
for pairwise comparison and all expressed as (x̄ ±s). The
group compared with the 2 µM shikonin group. With
Statistical Package for the Social Sciences (SPSS) 22.0
the increase of shikonin concentration, the percentage
software package was used to analyze the statistical
of cells in G0/G1 was increased significantly (p<0.05)
data in this study and the statistical results of p<0.05
in each group as shown in Table 2.
was considered that the difference was statistically

TABLE 1: CHANGES OF CELL ACTIVITY (x̄ ±s)

Cell activity (%)
Group Number of cases
12 h 24 h 48 h
0 µM shikonin group 6 100.00±0.01 100.00±0.01 100.00±0.01
2 µM shikonin group 6 96.65±1.42 a
92.71±1.55 a
73.11±1.26a
4 µM shikonin group 6 91.44±1.79ab 49.58±3.73ab 14.77±3.62ab
F 64.15 819.25 2325.9
p <0.001 <0.001 <0.001
Note: Compared with 0 µM shikonin group, p<0.05 and compared with 1 µM shikonin group, p<0.05
a b

Fig. 1: Cell activity changes in each group, ( ): 0 µM shikonin group; ( ): 2 µM shikonin group and ( ): 4 µM shikonin group

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TABLE 2: CHANGES OF CELL PERCENTAGE IN G0/G1 OF EACH GROUP (x̄ ±s)

Group Number of cases Percentage of cells in G0/G1
0 µM shikonin group 6 42.69±1.63
2 µM shikonin group 6 50.86±1.38a
4 µM shikonin group 6 57.23±0.22ab
F 207.43
p <0.001
Note: Compared with 0 µM shikonin group, ap<0.05 and compared with 1 µM shikonin group, bp<0.05

The percentage of senescent cells was significantly group as shown in Table 4.

increased (p<0.05) in the 2 µM shikonin group 48 h after
Expression levels of cyclin D1 and KDM2B were
the action of shikonin compared with that in the 0 µM
significantly decreased in the 2 µM shikonin group 48
shikonin group and the percentage of senescent cells in
h after the action of shikonin compared with the 0 µM
the 4 µM shikonin group was significantly increased
shikonin group while the expression levels of p-H2AX,
(p<0.05) compared with the 2 µM shikonin group. With
p53 and p21waf were significantly increased (p<0.05);
the increase of shikonin concentration, the percentage
and the expression levels of cyclin D1 and KDM2B
of senescent cells was increased significantly (p<0.05)
were significantly decreased in the 4 µM shikonin
in each group as shown in Table 3 and fig. 2.
group compared with the 2 µM shikonin group while
The ROS content was significantly increased (p<0.05) in the expression levels of p-H2AX, p53 and p21waf were
the 2 µM shikonin group 48 h after the action of shikonin significantly increased (p<0.05). With the increase of
compared with the 0 µM shikonin group and the percentage shikonin concentration, the expression levels of cyclin
of ROS content in the 4 µM shikonin group was significantly D1 and KDM2B were decreased significantly in each
increased (p<0.05) compared with the 2 µM shikonin group. group while the expression levels of p-H2AX, p53 and
With the increase of shikonin concentration, the ROS p21waf were increased significantly (p<0.05) as shown
content was decreased significantly (p<0.05) in each in fig. 3.

TABLE 3: CELL SENESCENCE IN EACH GROUPS (x̄ ±s)

Group Number of cases Percentage of senescent cells

0 µM shikonin group 6 10.04±0.09

2 µM shikonin group 6 49.74±2.68a

4 µM shikonin group 6 81.67±2.36ab

F 1816.55

p <0.001

Note: Compared with 0 µM shikonin group, ap<0.05 and compared with 1 µM shikonin group, bp<0.05

TABLE 4: ROS EXPRESSION LEVELS IN EACH GROUP (x̄ ±s)

Group Number of cases ROS content (%)

0 µM shikonin group 6 8.63±0.74

2 µM shikonin group 6 11.85±0.81a

4 µM shikonin group 6 28.97±2.67ab

F 258.21

p <0.001

Note: Compared with 0 µM shikonin group, ap<0.05 and compared with 1 µM shikonin group, bp<0.05

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Fig. 2: Cell senescence in each group, (A): 0 µM shikonin group; (B): 2 µM shikonin group and (C): 4 µM shikonin group

Fig. 3: Expression levels of cyclin D1, KDM2B, p-H2A.X, p53 and p21waf in each group, ( ): 0 µM shikonin group; ( ): 2 µM shikonin
group and ( ): 4 µM shikonin group

Lung cancer is one of the most common respiratory the growth of tumor cells and induce cell death[6]. The
malignancies clinically. In China, its morbidity and purpose of this study is to investigate the related roles
mortality rank first in malignant tumors. Among them, and mechanisms of shikonin in lung cancer.
non-small cell lung cancer accounts for about 80 % of
Cell senescence is a comprehensive manifestation of
lung cancer and it is a common type of lung cancer.
the decline and disorder of physiological function in
At present, surgical resection is still the main method
the degenerative period and it is an irreversible process.
for the treatment of lung cancer. However, the clinical
Some studies have found that initiating cell senescence
manifestations of lung cancer are often complex and
procedure will significantly improve the efficacy
diverse. In the early stage of lung cancer, because the
of anti-tumor drugs, which may be an important
clinical symptoms of most patients are not obvious, it
mechanism[7] to inhibit tumors. The main feature of
is difficult to attract the attention of patients. Therefore,
tumor cells is that the cells proliferate indefinitely
most of the patients have already been in the middle and
without differentiation, with some characteristics of the
late stage at the time of diagnosis. Some patients even
immortalization. More and more studies have found that
have experienced distant metastasis and missed the best
the cell immortalization has an important relationship
treatment time. Moreover, the prognosis of the patients
with tumorigenesis and development, which may be an
is poor and the 5 y survival rate is low. Therefore, it
important prerequisite for tumorigenesis, while the cell
is the main goal of the current treatment to find out a
senescence is a process[8,9] against cell immortalization.
therapy to alleviate the patients’ condition and improve
It can be seen that inducing tumor cell senescence may
their quality of life. As one of the effective ingredients
be an important way to inhibit tumors. Furthermore,
of Lithospermum, shikonin has been used for a long
some studies have found that cell cycle G0/G1 arrest
time in the treatment of burns, carbuncle, measles,
is also a prerequisite[10] for the cell senescence. Cell
yellow macula and sore throat, etc. In recent years,
cycle is the basic process of life activity between the
some scholars have found that the shikonin can inhibit
836 Indian Journal of Pharmaceutical Sciences July-August 2022
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cells and organism. The cell cycle plays an important Conflict of interests:
role in cell proliferation, differentiation, senescence and
death. When the cell cycle is abnormal, the abnormal The authors declared no conflicts of interest.
expression of cell division genes will make the cells
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