Invest Clin 64(4): 441 - 450, 2023 https://doi.org/10.54817/IC.v64n4a1

Correspondence author: Qun Zhao, Department of Cardiovascular Medicine (Group 1, 4th Treatment Area), Af-

filiated Hospital of Beihua University, Jilin, China. Email: zrx202102bh@163.com

Effects of focused ultrasound on human

cervical cancer HeLa cells in vitro.

Yanbin Liu1, Qun Zhao2, Panpan Liu3, Yanbin Li3, Li’an Yi4 and Haiping Yan4

1Department of Ultrasound Diagnosis, Affiliated Hospital of Beihua University, Jilin,

China.

2Department of Cardiovascular Medicine (Group 1, 4th Treatment Area), Affiliated

Hospital of Beihua University, Jilin, China.

3Department of Ultrasonic Medicine, Qingdao West Coast New Area Traditional Chinese

Medicine Hospital (Qingdao Huangdao District Traditional Chinese Medicine Hospital),

Qingdao, China.

4Department of Health Management Section, Qingdao West Coast New Area Traditional

Chinese Medicine Hospital (Qingdao Huangdao District Traditional Chinese Medicine

Hospital), Qingdao, China.

Keywords: focused ultrasound; cervical cancer; HeLa cell line; in vitro effects.

Abstract. Cervical cancer is the fourth most common malignant tumor

in women. Many studies have confirmed that early childbirth, prolificacy, HPV

infection, and smoking are some risk factors. This article explored the effects

of exposing human cervical cancer HeLa cells to different focused ultrasound

intensities in vitro. The study employed three groups of cells: 1- a high-intensity

treated group, 2- a low-intensity treated group, and 3- a control group. Results

showed that after 12 hours of focused ultrasound treatment, the growth inhibi-

tion rate of the low-intensity group was 55.6% higher than that of the control

group, and the growth inhibition rate of the high-intensity group was 41.2%

higher than that of the low-intensity group. Therefore, focused ultrasound had

a specific inhibitory effect on the growth of HeLa cells, and the higher the

intensity of focused ultrasound, the higher the inhibition rate on cancer cells.

In addition, the Cycle Threshold (Ct) values of the three groups of cells before

treatment were the same, but the Ct values after treatment had changed. The

Ct value of the low-intensity group was 18.1% lower than that of the control

group, and the Ct value of the high-intensity group was lower than that of the

low-intensity group by 27.8%, showing that focused ultrasound can effectively

reduce the activity of HeLa cells in vitro.

442 Liu et al.

Investigación Clínica 64(4): 2023

Efectos del ultrasonido focalizado sobre células HeLa de cáncer

cervical humano in vitro.

Invest Clin 2023; 64 (4): 441 – 450

Palabras clave: ultrasonido focalizado; cáncer de cuello uterino; línea celular HeLa;

efectos in vitro.

Resumen. El carcinoma de cuello uterino es el cuarto tumor maligno más

común en las mujeres. Muchos estudios han verificado que el parto prematu-

ro, la prolificidad, la infección por VPH y fumar son algunos de los factores de

riesgo. El propósito de este artículo fue investigar los efectos del tratamiento

con diferentes intensidades de ultrasonido focalizado sobre células HeLa de

cáncer de cuello uterino humano in vitro. Este estudio utilizó tres grupos de

células HeLa: 1- un grupo de tratamiento con alta intensidad, 2- un grupo de

tratamiento con baja intensidad y 3- un grupo control. Los resultados mostra-

ron que después de 12 horas de tratamiento con ultrasonido focalizado, la tasa

de inhibición del crecimiento del grupo de baja intensidad fue 55,6% más ele-

vada que la del grupo control y la tasa de inhibición del crecimiento del grupo

de alta intensidad fue 41,2% más elevada que la del grupo de baja intensidad.

Por lo tanto, el ultrasonido focalizado tiene un efecto inhibitorio sobre el cre-

cimiento de células HeLa, y cuanto mayor sea la intensidad del ultrasonido

focalizado, más elevada será la tasa de inhibición de las células cancerosas. Ade-

más, los valores del Umbral de Ciclos [Cycle Threshold (Ct)] de los tres grupos

de células eran los mismos antes del tratamiento, pero estos valores tuvieron

cambios evidentes después del tratamiento. El valor del Ct del grupo de baja

intensidad fue 18,1% inferior al del grupo de control y el valor del Ct del grupo

de alta intensidad fue 27,8% más bajo que el del grupo de baja intensidad; lo

que demuestra que el ultrasonido focalizado puede reducir la actividad de las

células HeLa in vitro.

Received: 23-08-2022 Accepted: 01-08-2023

INTRODUCTION

Cervical cancer is the most common

malignant tumor of the reproductive organs

in women. Based on the statistics, more

than 500,000 new cervical cancer cases oc-

cur yearly; about 5% of all new cancer pa-

tients and more than 80% live in developing

countries 1. Many studies have confirmed

that sexual disorders, oral contraceptives,

bisphenol A, early sexual life, nutritional

factors, premature birth, fertility, human

papillomavirus (HPV) infection, and smok-

ing contribute to the pathogenesis 2,3. Zhang

et al. investigated the relationship between

HPV16 E6 and E7 protein expression and

telomerase in cervical cancer carcinogen-

esis. The results showed that these proteins

and telomerase increase gradually with the

progression of cervical cancer 4. Sufficient

and improved screening programs to detect

this kind of cancer increase the knowledge

about its relation with HPV, reducing disease

cases in developed countries 5.

Patients may have no apparent symp-

toms in the early stage of this disease. With

Focused ultrasound on human cervical cancer HeLa cells in vitro 443

Vol. 64(4): 441 - 450, 2023

the disease progression, vaginal bleeding and

vaginal drainage, and even systemic failure

signs like anemia and cachexia in the later

stages may appear 6, which seriously threat-

en the lives and health of most women.

The common symptoms of cervical can-

cer are vaginal bleeding, vaginal discharge,

frequent urination, urgency, constipation,

swelling of the lower limbs, and abdominal

pain caused by the involvement of adjacent

tissues, organs, and nerves. In recent de-

cades, due to the wide application of cervi-

cal cytology screening technology, early di-

agnosis and treatment of cervical cancer and

precancerous lesions have become the main

reason for reducing morbidity and mortality,

thereby improving patient survival. A study

investigated the positive effect of using

WhatsApp (the internet messaging app) on

the health promotion of older women’s be-

havior for early detection of cervical cancer

through the visual acetic acid examination 7.

Focused ultrasound technology is a

high-tech that has gradually matured and

developed in recent years. Its basic principle

is to use ultrasound to have good perme-

ability inside the tissue and focus on iden-

tification. Through the computer control

system, the thermal effect, cavitation effect,

and mechanical effect eventually make the

target tumor cell degeneration and necrosis

to achieve the purpose of treatment3tissue

disintegration is also possible because of

the interaction between the distorted HIFU

bursts and either bubble cloud or boiling

bubble. Hydrodynamic cavitation is another

type of cavitation and has been employed

widely in industry, but its role in mechani-

cal erosion to tissue is not clearly known. In

this study, the bubble dynamics immediately

after the termination of HIFU exposure in

the transparent gel phantom was captured

by high-speed photography, from which the

bubble displacement towards the transducer

and the changes of bubble size was quantita-

tively determined. The characteristics of hy-

drodynamic cavitation due to the release of

the acoustic radiation force and relaxation

of compressed surrounding medium were

found to associate with the number of pulses

delivered and HIFU parameters (i.e. pulse

duration and pulse repetition frequency. In

recent decades, because focused ultrasound

has the advantages of non-invasiveness,

quick recovery after surgery, and less pain

for patients, it has been increasingly used to

treat various solid tumors, such as cervical

cancer, uterine fibroids, and pancreatic can-

cer 8. Many clinical studies have confirmed

that focused ultrasound can play a better

role in treating cervical cancer 9,10.

Imankulov et al. evaluated the feasibil-

ity of using high-intensity focused ultrasound

(HIFU) to treat tumors and proved that high-

intensity focused ultrasound can effectively

inhibit the growth of various tumor cells 11.

Hong et al. discussed the effect of HIFU irra-

diation on the apoptosis-related genes of hu-

man pancreatic cancer xenograft tumors. By

establishing a nude mouse-human pancreatic

cancer YY-1 cell xenograft model and HIFU ir-

radiation, the original TUNEL labeling meth-

od was used to detect the apoptosis rate of

tumor cells, and it was found that the tumor

cell apoptosis rate in the irradiated group was

higher 12. Yuan et al. reported that the mecha-

nism by which HIFU enhances anti-tumor

immunity has not been well elucidated, and

there is emerging evidence that miRNA plays

an essential role in the immune response 13.

Focused ultrasound for the treatment

of cervical cancer uses the directionality of

ultrasound, tissue penetration, and focusing

and uses special focusing equipment to fo-

cus ultrasound from the outside of the body

to the selected treatment area in the body14.

In some studies, focused ultrasound treat-

ment was recorded, and the average number

of treatments was eight times. Most patients

completed the follow-up. Among patients

who completed follow-up, survival rate statis-

tics were conducted every six months for the

24 months after treatment. The survival rate

in the 6th month was 100.00%; the survival

rate at the 12th month was 75.93%; the sur-

vival rate at the 18th month was 66.67%; the

444 Liu et al.

Investigación Clínica 64(4): 2023

survival rate at the 24th month was 55.56%.

These results show that focused ultrasound

can kill cancer tissues in a targeted manner

while preserving most normal tissues 15.

This article aimed to analyze the effect

of focused ultrasound on human cervical

cancer HeLa cells in vitro.

MATERIALS AND METHODS

Preparation of Experimental Materials

A medical university’s ultrasound en-

gineering institute provided human cervi-

cal cancer HeLa cells. The cultured HeLa

cells were randomly divided into control,

low-intensity, and high-intensity irradiation

groups. The control group was treated with

conventional interferon in vitro, the low-

intensity group was treated with focused ul-

trasound, and the high-intensity group was

treated with an intensity-focused ultrasound

instrument (Table 1).

The conventional cell culture method

The cryopreserved human cervical can-

cer HeLa cells were recovered in a culture

bottle containing 10% fetal bovine serum

RPM H-1640 nutrient solution, and a single-

cell suspension was prepared. Cell growth in-

hibition was determined by MTT colorimetry

(tetrazolium dye colorimetric assay).

The density of human cervical cancer

HeLa cells was adjusted to 3x103 cells/mL.

The MTT method was used to determine the

concentration, and the inhibition rate (R)

was calculated as follows:

Inhibition rate (R) = (1-experimental

group A value, control group A value) × 100%

The experiment was repeated three

times with the same method.

Focused ultrasonic field treatment

Logarithmically grown cells were taken

and digested with 0.25% trypsin. They were

centrifuged and washed with PBS solution

2~3 times. The cell density was adjusted to 2

x 104 cells/mL. The experimental group was

divided into low-dose and high-dose groups

according to the different intensities of fo-

cused ultrasound (0, 1000 and 3000 Watts/

cm2). A volume of 500 µL was taken out of

cell suspension from each group and placed

in the smallest electric chamber. The fixed

electric field intensity was 250 kV/cm, the

repetition frequency was 3 Hz, and the pulse

width was 800 ps. The control group did not

receive focused ultrasound treatment.

Observing the cell morphology under

an optical microscope

The cell density was adjusted to 3x104

cells/mL and inoculated into a 24-well culture

plate with a preset cover glass. Each well was

placed in an incubator for 1ml culture, and the

medium changed in the logarithmic growth

phase. After 72 hours, the coverslip was re-

moved and stained, and the cell morphology

was observed under an optical microscope.

Observing the cell morphology by

transmission electron microscopy (TEM)

The cells were inoculated in a culture

flask according to the above density. They

were collected, fixed before washing through

4% glutaraldehyde, fixed after washing with

1% acid, periodically dehydrated, and then

soaked with epoxy resin. They were then

embedded to make a fat mass and cut into

ultra-thin sections. The cell morphology was

observed with TEM.

Table 1

The condition and treatment of Hela cells in different groups.

Serial number Number of cells Passages Treatment

Control group 1.83×1045 Alpha interferon

Low-intensity group 1.75×1045 Low-intensity Focused ultrasound

High-intensity group 1.82×1045 High-intensity focused ultrasound

Focused ultrasound on human cervical cancer HeLa cells in vitro 445

Vol. 64(4): 441 - 450, 2023

Detecting cell apoptosis by the TUNEL

method

Cell slides were obtained according to

the method of observing cell morphology

under an optical microscope and cultured in

an incubator. After the logarithmic growth

phase, the medium was changed, and drugs

were added, according to the TUNEL meth-

od, which was utilized for detecting apoptot-

ic DNA fragmentation and identifying and

quantifying apoptotic cells.

Western blot analysis of BAX and Bcl-2

protein expression

After cell treatment, each group of cells

was incubated for 12 hours, suspended in PBS,

and counted. The 1x106 cells were pelleted by

centrifugation, lysed with 200 mL PMSF cell

lysis buffer for one hour, and then centrifuged

at 12000 g at 4 °C for 10 minutes. Bcl-2 and

BAX primary antibodies were added overnight

at 4 °C. The samples were washed three times

at room temperature in TBST for 10 minutes

each time. Quantity One ® 1-D image analysis

software (Bio-Rad) was used to analyze the

results. The absorbance of the blot was equal

to the average absorbance × area. The ratio

of BAX / Bcl-2 was equal to the absorbance of

the BAX band/Bcl-2 band.

Immunohistochemical staining (SP

method) was performed, and the steps of the

SP kit instructions were followed. PBS was

used instead of the primary antibody as a neg-

ative control, and Fuzhou Maxim Biological

Company provided photos of the positive pair.

Real-time Quantitative Polymerase Chain

Reaction (rtPCR) and Cycle Threshold

(Ct) Determination

Cycle Threshold (Ct) values were de-

termined using the real-time quantitative

polymerase chain reaction (rtPCR) method.

Total RNA was extracted from the control,

low-intensity, and high-intensity HeLa cell

samples before and after treatment using

the TRIzol reagent. The quality and concen-

tration of the extracted RNA were measured

using a NanoDrop spectrophotometer.

The extracted RNA was then reverse-tran-

scribed into cDNA using a reverse transcription

kit. The cDNA was used as the template for real-

time PCR amplification. The reaction mixture

included SYBR Green PCR Master Mix, forward

and reverse primers specific to the gene of in-

terest, and the cDNA template.

The cDNA was amplified by real-time

PCR using SYBR Green PCR Master Mix on

an Applied Biosystems 7500 Fast Real-Time

PCR system. The amplification conditions

were as follows: initial denaturation at 95 °C

for 10 min, followed by 40 cycles of denatur-

ation at 95 °C for 15 sec, annealing at 60 °C

for 1 min, and extension at 72 °C for 30 sec.

The Ct value indicates the number of

cycles required for the fluorescence signal to

surpass the threshold level, which is inversely

proportional to the amount of target nucleic

acid in the sample. Lower Ct values corre-

spond to higher levels of target nucleic acid.

The Primer Premier software designed

PCR primers specific for the reference gene

GAPDH, and the target gene was used. Rela-

tive expression levels of the gene of interest

were calculated using the 2-ΔΔCT method,

with GAPDH as the internal control. Fold

changes in gene expression were analyzed

using the 2-ΔΔCt method, and qRT-PCR was

performed in triplicate for each sample. The

Ct values of the control, low-intensity, and

high-intensity groups before and after treat-

ment were compared to evaluate the effect

of various treatments on HeLa cell activity.

Lower Ct values were indicative of higher

gene expression and cell activity.

Statistical processing

The IBM SPSS 18.0® statistical analy-

sis software was used for statistical process-

ing. All experiments were performed at least

three times. MTT results were analyzed

for the variance of repeated measurement

data, and laser scanning confocal and West-

ern blot detection was performed using the

ANOVA test. The difference was statistically

significant with p<0.05.

446 Liu et al.

Investigación Clínica 64(4): 2023

RESULTS

Comparison of the mortality of each

group of cells after focused ultrasound

treatment

The mortality of each group of cells was

measured using focused ultrasound treat-

ment of human cervical cancer HeLa cells at

three h, six h, 12 h, 24 h, and 48 h. The mor-

tality of cells in each group at 48 h showed

an upward trend with time, but the rising

speed of each group was different. It can be

seen that there is a difference between the

death rate of human cervical cancer HeLa

cells and the intensity of focused ultrasound

have a proportional relationship. In addition,

the cell death rate of HeLa cells in the same

treatment group reached a peak 12 hours af-

ter treatment, which was significantly higher

than other time points (P<0.01) (Table 2).

Comparison of the growth inhibition rate

of focused ultrasound

The growth inhibition of human cervical

cancer HeLa cells by focused ultrasound af-

ter different times was statistically analyzed.

It can be seen from the data in Fig. 1 that

focused ultrasound has a specific inhibitory

effect on the growth of HeLa cells, and the

higher the intensity of focused ultrasound,

the higher the inhibition rate on cancer cells.

After 12 hours of focused ultrasound treat-

ment, the growth inhibition rate of the low-

intensity group was 55.6% higher than that

of the control group, the growth inhibition

rate of the high-intensity group was 41.2%

higher than that of the low-intensity group,

and 72 hours after the focused ultrasound

treatment, the growth inhibition of the low-

intensity group was 55.6% higher than that of

the control group. The growth inhibition rate

of the high-intensity group was 11.1% higher

than that of the low-intensity group.

Western blot detection of BAX and Bcl-2

expression

It can be seen from the data in Fig. 2

that the expression of BAX protein increased

significantly after focused ultrasound treat-

ment. The expression of BAX in the low-in-

tensity group was 13.6% higher than that in

the control group, and the expression of BAX

in the high-intensity group was 2.1 higher

than that in the low-intensity group. The ex-

pression of Bcl-2 protein decreased after fo-

cused ultrasound treatment. The expression

of Bcl-2 in the low-intensity group was 15.9%

lower than that in the control group, and

the expression of Bcl-2 in the high-intensity

group was 51.7% lower than that of the low-

intensity group.

Changes in Ct values before and after

treatment of HeLa cells

Table 2

The percentage of mortality of different groups of cells at different time points.

Group 3 h 6 h 12 h 24 h 48 h

Control group 1.05±1.25 2.57±0.98 4.67±1.28 5.60±1.22 7.05±3.44

Low-intensity group 35.94±1.68 71.23±0.99 74.22±0.87 57.56±0.99 74.88±1.96

High-intensity group 56.94±3.68 83.24±0.25 85.23±0.45 85.68±2.97 85.88±1.01

Values are mean ± standard deviation.

Fig. 1. Comparison of the growth inhibition rate of

focused ultrasound on human cervical can-

cer HeLa cells after different times.

Focused ultrasound on human cervical cancer HeLa cells in vitro 447

Vol. 64(4): 441 - 450, 2023

This study recorded the Ct value chang-

es of three groups of human HeLa cells before

and after treatment. It can be seen from the

data in Fig. 3 that the Ct values of the three

groups of HeLa cells before treatment are

the same, but the Ct values after treatment

have apparent changes. After treatment, the

Ct value of the low-intensity group is 18.1%

lower than that of the control group. The Ct

value of the intensity group is 27.8% lower

than that of the low-intensity group, which

shows that focused ultrasound can effective-

ly reduce the activity of HeLa cells.

DISCUSSION

This study analyzed the effect of focused

ultrasound on human cervical cancer HeLa

cells in vitro, and it showed that. This study

showed that HIFU could be used to treat cer-

vical cancer. Specifically, the following three

points: first, this article uses a controlled ex-

periment to compare the in vitro effects of

low-intensity and high-intensity focused ul-

trasound. It accurately reflects the inhibitory

effect of focused ultrasound on HeLa cells.

Secondly, this article uses Western blot to de-

tect the expression of BAX and Bcl-2 proteins,

which can accurately reflect the changes in

protein expression in HeLa cells and the tu-

mor suppressor effect of focused ultrasound

at the protein level. Thirdly, MTT results for

repeated measurement data analysis of vari-

ance, laser scanning confocal, and Western

blot detection using ANOVA test to ensure

the experimental results are rigorous and

credible.

The research results show that focused

ultrasound has a particular inhibitory effect

on the growth of human HeLa cells, and the

higher the intensity of focused ultrasound,

the higher the inhibition rate on cancer

cells. After 12 hours of focused ultrasound

treatment, the growth inhibition rate of the

low-intensity group was 55.6% higher than

that of the control group, and the growth in-

hibition rate of the high-intensity group was

41.2% higher than that of the low-intensity

group. A study found that HIFU combined

with cisplatin facilitates tumor volume re-

duction and could be beneficial in treating

patients with cervical cancer 16. In addition,

Abel et al. reported that HIFU is a potentially

safe way to treat cervical cancer 17.

Bcl-2 is a member of the apoptotic gene

family. It is known that Bcl-2 is expressed

in solid tumors and is mainly located in the

cytoplasm and nucleus. Scientists speculate

that this may be related to the formation of

certain malignant tumors 18. In the related

studies of cervical cancer, although the ex-

pression of Bcl-2 has no apparent relationship

with tumor histological type, tumor stage, or

lymph node metastasis, the five-year survival

rate of Bcl-2-positive patients is significantly

higher than in BAX-positive patients 19.

In addition, BAX protein expression

was significantly increased after focused ul-

trasound treatment. The expression of BAX

in the low-intensity group was 13.6% higher

Fig. 2. Comparison of the results of Western blot

detection of BAX and Bcl-2 expression.

Fig. 3. Changes of Ct values before and after

treatment of HeLa cells.

448 Liu et al.

Investigación Clínica 64(4): 2023

than that in the control group, and the ex-

pression of BAX in the high-intensity group

was 2.1 times higher than that in the low-

intensity group, while the Bcl-2 protein after

focused ultrasound treatment, the expres-

sion level decreased. The expression level of

BAX in the low-intensity group was 15.9%

lower than that of the control group, and the

expression level of BAX in the high-intensity

group was 51.7% lower than that of the low-

intensity group.

According to the study results, the Ct

values of the three groups of HeLa cells be-

fore treatment were the same, but the Ct

values after treatment had changed. After

treatment, the Ct values of the low-intensity

group were 18.1% lower than those of the

control group, and the Ct value of the high-

intensity group was 27.8% lower than the

low-intensity group, showing that focused

ultrasound can effectively reduce the activ-

ity of HeLa cells. In addition, focused ultra-

sound has the highest proportion in treating

pancreatic cancer, reaching 51.4%, followed

by bone tumors, accounting for 21.1%, while

cervical cancer only accounts for 3% (Fig. 4)

20,21. It can be seen that focused ultrasound

has a more favorable prospect for treating

cervical cancer.

In summary, this article proposed fo-

cused ultrasound as a new treatment meth-

od for cervical cancer. This study briefly in-

troduced focused ultrasound as a treatment

method, studied the in vitro effects of focused

ultrasound on HeLa cells, and analyzed the

inhibitory effect of focused ultrasound on

cancer cells. The research results show that

focused ultrasound has a specific inhibitory

effect on the growth of HeLa cells, and the

higher the intensity of focused ultrasound,

the higher the inhibition rate on cancer cells.

ACKNOWLEDGMENTS

We thank Dr. Humberto Martinez for

this manuscript’s quality enhancement and

language-native editing.

Funding

This study was not funded.

Conflict of interests

All of the authors had no personal, fi-

nancial, commercial, or academic conflicts

of interest separately.

Authors’ ORCID numbers

• Yanbin Liu (YLiu):

0000-0003-4511-061X

• Qun Zhao (QZ):

0000-0002-1119-503X

• Panpan Liu (PL):

0000-0002-8796-2426

• Yanbin Li (YLi):

0000-0001-5499-8986

• Li’an Yi (LY):

0000-0003-2994-7233

• Haiping Yan (HY):

0000-0001-7449-2616

Authors’ Contribution

YLiu: Manuscript editing, revising and

final approval of manuscript. QZ: Manuscript

editing, revising and final approval of manu-

Fig. 4. Application of focused ultrasound in the

treatment of solid tumors and its propor-

tion.

Focused ultrasound on human cervical cancer HeLa cells in vitro 449

Vol. 64(4): 441 - 450, 2023

script. PL: Administrative support. YLi: Con-

ception and design, Collection and assembly

of data. LY: Provision of study materials or pa-

tients. HY: Data analysis and interpretation.

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