Invest Clin 65(3): 267 - 278, 2024 https://doi.org/10.54817/IC.v65n3a01

Corresponding authors: Shaoqun Liu and Chang Su. Department of Gastrointestinal Surgery, Minhang Hospital

Affiliated to Fudan University, Xinzhuang Town, Shanghai 201199, China. E-mail: liushaoqun@fudan.edu.cn

E-mail: surgeon_suchang@fudan

Dihydroartemisinin, an artemisinin

derivative, reverses oxaliplatin resistance

in human colorectal cancer cells

by regulating the SIRT3/PI3K/AKT

signalling pathway.

Xiaodong Shen, Chencheng Shi, Ming Lei, Rongjian Zhou, Shaoqun Liu and Chang Su

Department of Gastrointestinal Surgery, Minhang Hospital Affiliated to Fudan

University, Shanghai, China.

Keywords: dihydroartemisinin; SIRT3; oxaliplatin; colorectal cancer.

Abstract. Dihydroartemisinin (DHA), a derivative of artemisinin, has been

shown to act as a chemosensitizer of various cancer chemotherapeutic agents

both in vitro and in vivo. However, in colorectal cancer (CRC), no study has fo-

cused on the effect of DHA on oxaliplatin (L-OHP) resistance. Our study aimed

to examine the effectiveness of DHA in reversing the resistance of human CRC

cells to L-OHP, as well as its underlying molecular mechanisms. LoVo cells were

purchased from ATCC, while LoVo/L-OHP cells were obtained by exposing LoVo

cells to progressively increasing concentrations of L-OHP. LoVo/L-OHP were

treated with various concentrations of DHA, and cell apoptosis ratio and vi-

ability were assessed by flow cytometry and CCK-8. Our results showed that

DHA treatment remarkably decreased the viability of LoVo/L-OHP cells and

increased the apoptosis ratio. As the mechanism of action, we found that DHA

enhanced the expression of Sirtuin 3 (SIRT3) and suppressed the phosphati-

dylinositol 3-kinase (PI3K)/AKT signalling cascade. Silencing of SIRT3 reversed

the effect of DHA on cell apoptosis and viability by activating the PI3K/AKT

axis in LoVo/L-OHP cells. Overall, our study found that DHA has the ability to

counteract L-OHP resistance in LoVo/L-OHP cells through the modulation of

the SIRT3/PI3K/AKT signalling pathway, suggesting a new research target for

CRC treatment.

268 Shen et al.

Investigación Clínica 65(3): 2024

La dihidroartemisinina, un derivado de la artemisinina, revierte

la resistencia al oxaliplatino en células humanas de cáncer

colorrectal mediante la regulación de la vía de señalización

SIRT3/PI3K/AKT.

Invest Clin 2024; 65 (3): 267 – 278

Palabras clave: dihidroartemisinina; SIRT3; oxaliplatino; cáncer colorrectal.

Resumen. Se ha demostrado que la dihidroartemisinina (DHA), un deri-

vado de la artemisinina, actúa como quimiosensibilizador de diversos agentes

quimioterapéuticos contra el cáncer tanto in vitro como in vivo. Sin embargo,

en el cáncer colorrectal (CCR), ningún estudio se ha centrado en el efecto del

DHA sobre la resistencia al oxaliplatino (L-OHP). El objetivo de nuestro estu-

dio era examinar la eficacia del DHA para revertir la resistencia de las células

humanas de CCR al L-OHP, así como sus mecanismos moleculares subyacentes.

Las células LoVo se adquirieron a ATCC, mientras que las células LoVo/L-OHP

se obtuvieron exponiendo las células LoVo a concentraciones progresivamente

crecientes de L-OHP. Las células LoVo/L-OHP se trataron con diversas con-

centraciones de DHA, y el índice de apoptosis celular y la viabilidad se evalua-

ron mediante citometría de flujo y CCK-8. Nuestros resultados mostraron que

el tratamiento con DHA disminuía notablemente la viabilidad de las células

LoVo/L-OHP y aumentaba el índice de apoptosis. Desde el punto de vista de

su mecanismo de acción, se observó que el DHA potenciaba la expresión de la

sirtuina 3 (SIRT3) y suprimía la cascada de señalización fosfatidilinositol 3-ci-

nasa (PI3K)/AKT. El silenciamiento de SIRT3 revirtió el efecto del DHA sobre la

apoptosis y la viabilidad celular a través de la activación del eje PI3K/AKT en las

células LoVo/L-OHP. En conjunto, nuestro estudio descubrió la capacidad del

DHA para contrarrestar la resistencia a L-OHP en células LoVo/L-OHP a través

de la modulación de la vía de señalización SIRT3/PI3K/AKT, lo cual sugiere una

nueva foco de investigación para el tratamiento del CCR.

Received: 25-07-2023 Accepted: 27-09-2023

INTRODUCTION

According to the 2020 GLOBOCAN sta-

tistics on the incidence and mortality rates

of 36 types of cancer worldwide, colorectal

cancer (CRC) ranks third in terms of inci-

dence (10.0%) but second in terms of mor-

tality rate (9.4%) 1. Chemotherapy based on

oxaliplatin (L-OHP) is a crucial component

of the comprehensive treatment for CRC,

which can prolong the survival of patients

and improve their quality of life 2-4. How-

ever, primary and/or acquired resistance to

L-OHP remains a significant factor limiting

the effectiveness of CRC treatment 5,6. There-

fore, it is urgent to clarify the mechanisms

of L-OHP resistance in CRC and to find cor-

responding resistance reversal agents or

chemosensitizers.

Previous studies have demonstrated

that the mechanisms of resistance to plati-

num-based chemotherapy drugs may be re-

Dihydroartemisinin reverses oxaliplatin resistance in colorectal cancer 269

Vol. 65(3): 267 - 278, 2024

lated to the abnormal regulation of signal-

ling networks within tumor cells caused by

the sustained action of drugs. For example,

abnormalities in signalling pathways such as

PI3K/AKT and NF-κB give the cells strong

anti-apoptotic abilities, leading to resistance

to chemotherapy drugs 7,8. Sirtuin 3 (Sirt3)

plays a crucial role in controlling physiologi-

cal and pathological processes, such as regu-

lating inflammation, oxidative stress, cell

proliferation, differentiation, and apoptosis.

This protein belongs to the sirtuin protein

family, known for its diverse biological func-

tions 9-11. Research has found that SIRT3 can

cause mutant p53 to be deacetylated, lead-

ing to cancer cell apoptosis and increased

sensitivity to chemotherapeutic drugs 12.

Other evidence also suggested that SIRT3

could improve disease conditions by regu-

lating apoptosis and utilizing the PI3K/Akt

pathway in various diseases 13,14. Dihydroarte-

misinin (DHA) is a derivative of artemisinin,

and there is evidence that DHA can act as

a chemosensitizer for various cancer chemo-

therapeutic drugs both in vitro and in vivo15.

The latest research has found that DHA can

alleviate acute and chronic pain by increas-

ing the activity of SIRT3 16. However, in CRC,

no study has focused on the effect of DHA on

L-OHP resistance through the SIRT3/PI3K/

AKT axis.

Based on previous studies, we hypoth-

esize that DHA may affect the resistance of

human CRC cells to L-OHP by regulating the

SIRT3/PI3K/AKT axis. Our study aimed to

examine the effectiveness of DHA in revers-

ing the resistance of human colorectal can-

cer cells to L-OHP, as well as its underlying

molecular mechanisms.

METHOD

Cell culture and induction of L-OHP

resistance

Human CRC cells (LoVo, not resistant

to L-OHP) were obtained from the American

Type Culture Collection (CCL-229, ATCC)

in Manassas, Virginia. The human L-OHP-

resistant CRC cells (LoVo/L-OHP, resistant

to L-OHP) were obtained by exposing LoVo

cells to progressively increased concentra-

tions of L-OHP 17. The initial concentration

of L-OHP was 5 μM, and cells were exposed

to L-OHP for two days in each cycle, followed

by recovery in L-OHP-free culture medium.

The same concentration of L-OHP was re-

peated for three cycles. After completing

three cycles at the same concentration of

L-OHP stimulation, the dose was escalated.

This procedure was repeated until the final

concentration of L-OHP reached 70 μM.

Only cells that remained resistant to L-OHP

after being cultured in an L-OHP-free me-

dium for at least six months were used for

subsequent experiments.

The LoVo/L-OHP and LoVo cells were

cultured at 37°C and 5% CO2 in RPMI-1640

medium (Thermo Fisher Scientific, Inc.,

MA, USA) that was supplemented with 10%

Gibco® fetal bovine serum (FBS) and 100

μg/mL penicillin-streptomycin. Cells were

treated with PI3K inhibitor LY294002 (1μM)

for 24 hours to inhibit PI3K signaling.

Preparation of Dihydroartemisinin

Solution

A stock solution of dihydroartemisinin

(obtained from Puyi Biological, Nanjing,

China) was prepared by dissolving it in a

small quantity of dimethyl sulfoxide (DMSO,

procured from Sigma-Aldrich, USA). The

resulting stock solution was formulated to

have a concentration of either 100 or 300

nmol/L, which was sonicated and stored

at 4°C. In subsequent experiments, all cell

samples were treated with 30 μM L-OHP or

an equivalent amount phosphate-buffered

saline (PBS) for treatment.

Cell transfection

The siRNA targeting the SIRT3 gene (si-

SIRT3#1, #2, #3) and the negative control

(NC) were procured from GeneChem Corp.

for cell transfection experiments.

270 Shen et al.

Investigación Clínica 65(3): 2024

According to the manufacturer’s in-

structions, the LoVo/L-OHP cells were trans-

fected with 5 nM of the aforementioned si-

RNA or negative controls. Transfection was

carried out by using the lipofectamine 3000

(Invitrogen) reagent, following the pre-

scribed protocol, while incubating the cells

in a serum-free Opti-MEM medium. After 48

hours of transfection, the transfection effi-

ciency was evaluated through RT-qPCR.

Cell Counting Kit-8 (CCK-8)

LoVo, LoVo/L-OHP, and LoVo/L-

OHP+DHA (100nmol/L or 300nmol/L)

cells were treated with different concentra-

tions of OHP (5μM-70μM) 17,18. Cells were

cultured into 96-well plates at 5×103 cells/

well density. After incubating the cells with

CCK-8 reagent (10μL, Sangon) for 2 hours,

the absorbance at 450 nm was determined

on a microplate reader (Thermo Fisher Sci-

entific, MA, USA). The half-maximal inhibi-

tory concentration (IC50) of L-OHP was de-

termined in LoVo and LoVo/L-OHP cells 19.

Reverse transcription-quantitative

polymerase chain reaction (RT-qPCR)

RT-qPCR was used to determine the ex-

pression of PI3K, SIRT3 and AKT in the cells.

We utilized the RNAiso plus kit (procured

from Takara, Japan) to obtain RNA from

cells and subsequently performed reverse

transcription into cDNA using the Prime-

ScriptTM one-step qRT-PCR kit (obtained

from Takara, Dalian, China). For RT-qPCR,

we employed the SYBR Premix ExTaq (TaKa-

Ra) and the ABI PRISM7300 Sequence De-

tection System (from Applied Biosystems).

Primer sequences are shown in Table 1. GAP-

DH served as an internal control. The mRNA

expressions were calculated by the 2-ΔΔCt

method.

Western blot

Total proteins were extracted from the

cell lysates and quantified using the Pierce™

BCA Protein Assay Kit (Thermo Scientific).

Then, 20 μg of the extracted proteins were

separated by SDS-PAGE and transferred onto

PVDF membranes. The PVDF membranes

were then incubated with a primary antibody

at 4˚C overnight. After washing, the mem-

branes were incubated with Goat Anti-Rab-

bit IgG H&L secondary antibody (ab96899,

1/1000) at 37˚C for 45 minutes. The prima-

ry antibodies included the SIRT3 antibody

ab217319, 1/1000, Abcam), PI3K antibody

(ab140307, 1/2000, Abcam), AKT antibody

(ab8805, 1/1000, Abcam), bcl-2 antibody

(ab32124, 1/1000, Abcam), Bax antibody

(ab32503, 1/1000, Abcam), cleaved cas-

pase-3 antibody (ab32042, 1/5000, Abcam),

P-gb (ab202976, 1/1000, Abcam), GAPDH

antibody (ab8245, 1/1000, Mybiosource),

and GAPDH was served as an internal con-

trol. We utilized the Pierce ECL Western

Blotting Substrate to visualize the protein

bands, which was procured from Pierce in

Shanghai, China. The membranes were de-

veloped and imaged using a chemilumines-

cence imaging system.

Flow cytometry for cell apoptosis

Cells were incubated at 37°C with 5%

CO2. After the incubation of 24 h, LoVo/L-

OHP cells were collected into 1.5 mL tubes

with annexin-FITC and propidium iodide

(PI) regents, followed by incubation for 10

min. To detect cell apoptosis, 200 μL PI was

supplemented with 1 mL phosphate buffer

Table 1

The sequences of all constructs.

Name Sequence

SIRT3 Forward 5’-CCCCAAGCCCTTTTTCACTTT-3’

Reverse 5’-CGACACTCTCTCAAGCCCA-3’

PI3K Forward 5’-TCTAAACCCTGCTCATC-3’

Reverse 5’-CTTGCCGTAAATCATCCC-3’

AKT Forward 5’-CTACAACCAGGACCATGAGAAG-3’

Reverse 5’-ACACGATACCGGCAAAGAAG-3’

GAPDH Forward 5′-CACCCACTCCTCCACCTTTG-3′

Reverse 5′-CCACCACCCTGTTGCTGTAG-3′

Dihydroartemisinin reverses oxaliplatin resistance in colorectal cancer 271

Vol. 65(3): 267 - 278, 2024

solution (PBS) into the flow tube. Then, the

apoptotic cells were measured using flow cy-

tometry (Beckman Coulter).

Statistical analysis

The data is presented as mean ± SD,

and we utilized one-way ANOVA followed by

a Tukey post hoc test for statistical compari-

sons. For significance, a p-value less than

0.05 was considered.

RESULTS

DHA inhibited the proliferation of LoVo/

L-OHP cells and induced their apoptosis

We first treated LoVo/L-OHP cells with

different concentrations (100 nmol/L or 300

nmol/L) of DHA then used the CCK-8 assay to

detect the IC50 of all cells. As shown in Fig.

1-A, the IC50 of LoVo/L-OHP cells to L-OHP

remarkably declined (p<0.05) after DHA

treatment. In the next experiment, we treat-

ed LoVo/L-OHP cells with 30μM L-OHP or an

equal amount of phosphate-buffered saline

(PBS) and investigated the changes in cell vi-

ability of LoVo/L-OHP cells after DHA treat-

ment. Our findings demonstrated a notable

decline in cell viability of LoVo/L-OHP cells

following DHA treatment (p<0.05, as de-

picted in Fig. 1-B). Moreover, flow cytometry

analysis substantiated a marked rise in apop-

totic cells in LoVo/L-OHP cells when treated

with 300 nmol/L DHA (p<0.05, as illustrated

in Fig. 1-C). Furthermore, compared with the

LoVo/L-OHP group, the expression of cleaved

caspase-3 protein and the Bax/Bcl-2 ratio in

the L-OHP +300 nmol/L DHA and L-OHP

+100nmol/L DHA groups were remarkably

increased, while the expression of P-gb pro-

tein was markedly decreased (p<0.05, Fig.

1-D). In the subsequent experiments, cells

were treated with 300 nmol/L of DHA.

The expression of SIRT3, PI3K, and AKT

in LoVo/L-OHP cells after DHA treatment

To further investigate the expression of

SIRT3, PI3K, and AKT in LoVo/L-OHP cells

after DHA treatment, we examined the ex-

pression of SIRT3, PI3K, and AKT in each

group of cells using WB and PCR. As shown in

Figs 2 A-B, our results indicated a significant

decrease in SIRT3 expression level, whereas

PI3K and AKT expression levels were signifi-

cantly elevated in LoVo/L-OHP cells com-

pared to the LoVo group (p<0.05). After ad-

ministering L-OHP to LoVo/L-OHP cells and

subsequently treating them with 300 nmol/L

DHA, we observed a noteworthy upsurge

in the protein and mRNA levels of SIRT3 as

compared to the LoVo/L-OHP+L-OHP group

(p<0.05). Furthermore, we observed a reduc-

tion in expression levels of PI3K and AKT in

cells following DHA treatment (p<0.05).

Silencing SIRT3 promoted L-OHP

resistance in LoVo/L-OHP cells after DHA

treatment through the PI3K/AKT pathway

To validate the role of the SIRT3/PI3K/

AKT signalling pathway in L-OHP resistance

of LoVo/L-OHP cells after DHA treatment,

we transfected cells with si-SIRT3#1, si-

SIRT3#2, si-SIRT3#3, and si-NC and mea-

sured the knockdown efficiency of SIRT3.

As shown in Figs. 3 A-B, in LoVo/L-OHP

cells after DHA treatment, si-SIRT3#1,

si-SIRT3#2, and si-SIRT3#3 all markably

reduced the expression of SIRT3, and we

selected si-SIRT3#2 with the highest knock-

down efficiency for subsequent experiments.

In addition, we also used the PI3K inhibitor

LY294002 to inhibit the PI3K/AKT path-

way and divided the cells into groups: conc-

trl (LoVo/L-OHP+L-OHP+DHA), si-NC

(conctrol+si-NC), si-SIRT3 (conctrol+si-

SIRT3), and si-SIRT3+LY294002 (conctrol+

si-SIRT3+LY294002). As shown in Fig. 3-C,

compared with the si-NC group, silencing

SIRT3 remarkably increased cell viability

and reduced the proportion of cell apopto-

sis (p<0.05). However, the addition of the

PI3K agonist LY294002 reversed this effect.

Compared with the si-SIRT3 group, the si-

SIRT3+LY294002 group had significantly

reduced cell viability and increased apopto-

sis (Fig. 3-D).

272 Shen et al.

Investigación Clínica 65(3): 2024

Effects of SIRT3 knockdown on relevant

proteins in LoVo/L-OHP cells after DHA

treatment

Finally, we used WB to measure the

protein expression of the SIRT3/PI3K/AKT

signalling pathway proteins, the drug-resis-

tant protein P-gb, and the apoptosis-related

proteins cleaved caspase-3, Bax and Bcl-2. As

illustrated in Fig. 4, our data demonstrated

that transfection with si-SIRT3 resulted in a

notable decline in SIRT3 protein expression

and increased protein expression of PI3K

and AKT (p<0.05).

Fig. 1. DHA inhibited the proliferation of LoVo/L-OHP cells and induced their apoptosis. (A) The IC50 of

all groups of cells were detected by CCK-8 assay. (B) Cell viability of all groups of cells was detected

by CCK-8 assay. (C) The apoptosis rate of all groups of cells was analyzed by flow cytometry. (D) The

expression of P-gp protein related to drug resistance and apoptosis-related proteins, cleaved caspase-3

protein, Bax, and Bcl-2, was detected by Western blot. Continuous data were compared using ANOVA.

*p<0.05 compared with the L-OHP group. #p<0.05 compared with the LoVo/L-OHP group.

Dihydroartemisinin reverses oxaliplatin resistance in colorectal cancer 273

Vol. 65(3): 267 - 278, 2024

On the other hand, treatment with

PI3K inhibitor LY294002 remarkably re-

duced the protein expression levels of PI3K

and AKT (p<0.05). Silencing SIRT3 marked-

ly enhanced the expression of P-gb while re-

ducing the protein expression of cleaved cas-

pase-3 and the ratio of Bax/Bcl-2. However,

the PI3K inhibitor LY294002 reversed this

effect. In the si-SIRT3+LY294002 group, the

protein expression of P-gb decreased, while

the expression of cleaved caspase-3 and the

ratio of Bax/Bcl-2 significantly increased

(p<0.05).

DISCUSSION

For researchers, the mechanism of L-

OHP resistance in CRC is still elusive. De-

spite many molecular medicine studies to

explain the resistance mechanisms of CRC,

it is challenging to distinguish genuinely

effective targets for regulating resistance.

In addition, most existing multidrug resis-

tance reversal agents have issues such as

incomplete resistance reversal, high tech-

nical requirements, and significant adverse

reactions 20, 21. Therefore, finding new sensi-

tizers to combine with L-OHP to treat CRC

patients is still necessary. We demonstrated

that DHA could inhibit the L-OHP resistance

in LoVo human colorectal cancer cells by

regulating the SIRT3/PI3K/AKT axis.

Studies conducted in the past have dem-

onstrated that DHA can hinder the growth

of cancer cells and trigger apoptosis in pan-

creatic cancer, ovarian cancer, and colorec-

tal cancer. An added advantage of DHA is its

low toxicity towards normal tissue cells 22,

23. In addition, DHA also has a killing effect

on multidrug-resistant tumor cells 24. In our

study, we treated CRC-resistant cells LoVo/

L-OHP with different concentrations of DHA

and found that the viability of LoVo/L-OHP

cells marked decrease, and the proportion

of apoptotic cells increased significantly af-

ter DHA treatment. This phenomenon was

Fig. 2. The expression of SIRT3, PI3K and AKT in LoVo/L-OHP cells after DHA treatment. The SIRT3,

PI3K and AKT expression in each group were measured by WB (A) and PCR (B). ANOVA was used to

compare the groups. #p<0.05 compared with the LoVo/L-OHP group. *p<0.05 compared with the

LoVo/L-OHP+L-OHP group.

274 Shen et al.

Investigación Clínica 65(3): 2024

more pronounced in the group with higher

concentrations of DHA, indicating that DHA

can inhibit the resistance of LoVo/L-OHP to

L-OHP. Similar results have been obtained in

other cancers. In non-small-cell lung cancer

(NSCLC), DHA can eliminate radiation re-

sistance of NSCLC cell lines and xenograft

models by inhibiting the TGF-β, PI3K/Akt,

and STAT3 signalling pathways, and enhance

the anti-tumor effect of radiotherapy 25. In

breast cancer, DHA regulates the STAT3/

DDA1 axis to inhibit the proliferation of

breast cancer tumor cells 26. In addition, Yao

et al. confirmed that DHA can effectively sen-

sitize CRC cells to 5-fluorouracil (5-FU) by

mediating ROS-induced apoptosis 27. Wang

et al. also showed that DHA inhibits the char-

acteristics of cancer stem-like cells (CSLCs)

Fig. 3. Silencing SIRT3 promotes L-OHP resistance in LoVo/L-OHP cells after DHA treatment through

the PI3K/AKT pathway. The expression of SIRT3 was measured by WB (A) and PCR (B). (C) Cell via-

bility of all groups of cells was detected by CCK-8 assay. (D) The apoptosis rate of all groups of cells

was analyzed by flow cytometry. ANOVA was used to compare the groups. *p<0.05 vs si-NC group.

#p<0.05 vs si-SIRT3 group.

Dihydroartemisinin reverses oxaliplatin resistance in colorectal cancer 275

Vol. 65(3): 267 - 278, 2024

in CRC through the AKT/mTOR signalling

pathway 28. These results suggested that

DHA can be a sensitizing cancer treatment

agent.

The protein and mRNA levels of SIRT3

were enhanced in LoVo/L-OHP cells after

treatment with DHA, indicating that SIRT3

may be involved in the sensitization of LoVo/

L-OHP by DHA. Cao et al. found that acti-

vation of SIRT3 inhibits cisplatin resistance

in lung cancer cells and reduces the prolif-

eration and invasion of lung cancer cells 29.

Another research showed that the activity of

SIRT3 in colon cancer cells can be enhanced

by nitric oxide synthase (NOS), which helps

to promote cell apoptosis 30. To study the

mechanism of action, we silenced SIRT3 and

found that the viability of LoVo/L-OHP cells

improved, and the proportion of apoptotic

cells and apoptotic proteins cleaved cas-

pase-3 and the ratio of bax/bcl-2 decreased

significantly, indicating that the knockdown

of SIRT3 affected the resistance of LoVo/L-

OHP cells to L-OHP after DHA treatment.

Furthermore, we confirmed that silencing

SIRT3 enhanced the expression of PI3K and

AKT in LoVo/L-OHP cells. The aberrant acti-

vation of the PI3K/AKT pathway acts against

chemotherapy-induced apoptosis by increas-

ing the expression of anti-apoptotic genes

(bcl-2 and XIAP) and reducing the expres-

sion of pro-apoptotic genes (bax) 31. In our

study, we inhibited the PI3K/AKT axis with

LY294002 and found that the viability of

LoVo/L-OHP cells decreased while apopto-

sis increased. This also reversed the effect

of knocking down SIRT3 on the viability and

apoptosis of LoVo/L-OHP cells. Zhang et al.

32 found that inhibiting the PI3K/AKT signal-

ling pathway in CRC cells can inhibit cell

proliferation and activate apoptosis, thereby

reversing oxaliplatin resistance, consistent

with our research results.

Limitation

Our research also has some limitations.

Whether other genes regulate SIRT3 is not

clear. We also did not perform in vivo experi-

ments to confirm the apoptosis results. All

these need further studies to illustrate.

Our study found that DHA could inhibit

the L-OHP resistance in LoVo human colorec-

tal cancer cells by regulating the SIRT3/

PI3K/AKT axis. The results might provide a

new integrated treatment approach for CRC

treatment in clinical practice.

Ethics approval and consent to participate

The ethic approval was obtained from

the Ethic Committee of The Minhang Hos-

pital, Affiliated with Fudan University, and

Fig. 4. Effects of SIRT3 knockdown on relevant proteins in LoVo/L-OHP cells after DHA treatment. The

protein expression of SIRT3, PI3K, AKT, P-gb, cleaved caspase-3, Bax and Bcl-2 were measured by WB.

ANOVA was used to compare the groups. *p<0.05 vs si-NC group. #p<0.05 vs si-SIRT3 group.

276 Shen et al.

Investigación Clínica 65(3): 2024

written informed consent was obtained from

all patients.All of the authors have Consent-

ed to publish this research.

Availability of data and materials

The data are free to access and avail-

able upon request.

Competing interests

All authors declare no conflict of interest.

Funding

Project of Health Commission of

Minhang District, Shanghai, Grant No.

2021MW23.

ORCID number of authors

• Xiaodong Shen:

0009-0000-0520-8896

• Chencheng Shi:

0009-0008-1369-2946

• Ming Lei:

0009-0000-8480-197X

• Rongjian Zhou:

0009-0007-5598-0834

• Shaoqun Liu:

0000-0002-2016-0947

• Chang Su:

0009-0004-6481-2638

Authors’ contributions

Each author has made an important sci-

entific contribution to the study and assist-

ed with drafting or revising the manuscript.

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