Role of Endoplasmic Reticulum Stress in Melatonin-induced Apoptosis and Inhibition of Invasion and Migration in Adrenocortical Carcinoma Cells
Qingsun Lai1,2,1, Sheng Su2,1, Peichun He1, Haiyan Yang1, Zhenxing Huang1, Decheng Lu1, Zuojie Luo1,*
1 Department of Endocrinology, The First Affiliated Hospital of Guangxi Medical University, 530021 Nanning, Guangxi, China
2 Department of General Practice, Liuzhou Workers’ Hospital, 545007 Liuzhou, Guangxi, China
*Correspondence: luozuojie@gxmu.edu.cn (Zuojie Luo)
These authors contributed equally.
Published: 20 November 2024
Background: Melatonin, a hormone synthesized by the pineal gland and released into the blood, seems to have anti-tumor proper- ties. However, the mechanisms of the anti-cancer effect of melatonin are largely unknown. This study investigated the anti-tumor activity of melatonin in adrenocortical carcinoma (ACC) and analyzed its molecular mechanisms.
Methods: Different concentrations of melatonin were added to ACC cells in vitro and in vivo. Cell viability was appraised via Cell Counting Kit-8 (CCK-8) assay, cell migration and invasion were appraised via wound healing assay and transwell assay, and cell apoptosis was appraised via flow cytometry. The levels of nuclear factor kappa B (NF-KB)/mitogen-activated protein kinase (MAPK) pathway proteins (c-Jun N-terminal kinase (JNK) and p38) and endoplasmic reticulum stress-related proteins (C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78)) were appraised via western blot.
Results: Melatonin reduced the proliferation rate, migration rate, and invasion rate of ACC cells, and significantly increased apoptosis of ACC cells in contrast with the Control Check (CK) group. Moreover, melatonin intervention reduced NF-KB/MAPK signal routing (JNK and p38) and endoplasmic reticulum stress (CHOP and GRP78). Treatment with the NF-KB/MAPK pathway inhibitor NF-KB/MAPK-IN-1 (3.48 uM) enhanced the inhibitory effects of melatonin on the activity of ACC cells and increased apoptosis. The subcutaneous tumor model (SW-13) in nude mice further confirmed that melatonin induced apoptosis of ACC cells by reducing endoplasmic reticulum stress, and NF-KB/MAPK signal routing was involved in this effect. Conclusion: Melatonin induces apoptosis of ACC cells by reducing endoplasmic reticulum stress, and this effects was may be related to the NF-KB/MAPK signal routing. Melatonin may be an effective anti-tumor agent and have great potential as an adjuvant therapy in the future.
Keywords: adrenocortical carcinoma; melatonin; endoplasmic reticulum stress; NF-KB/MAPK; cell apoptosis
Introduction
Adrenocortical carcinoma (ACC), a malignant tumor that occurs in the adrenal cortex, is characterized by high malignancy and poor prognosis [1], the annual incidence of ACC in the population is about 0.7-2.0 cases/million peo- ple, accounting for 0.2% of cancer deaths [2]. ACC can occur in any age group, but there are two peak ages of on- set, namely, 1-6 years old in childhood and 40-50 years old in middle age. In the early stage of ACC, owing to the lack of specific clinical manifestations and characteristics, about 70% of ACC patients are diagnosed at stages III and IV of the disease [3], in patients with advanced ACC, most cases have local and distant invasion and metastasis; for pa- tients with stage IV ACC, the 5-year survival rate is only 6%-13% [4]. At present, there is no effective treatment for advanced ACC. Surgery, radiotherapy, and chemother-
apy have not shown good clinical efficacy. With the emer- gence of second-generation and third-generation sequenc- ing, genes closely related to the occurrence and develop- ment of ACC have been discovered. However, no effec- tive new targets or new drugs for ACC treatment have been found [5]. Therefore, an important focus of research is the development of effective drug therapy for ACC.
Melatonin is a bioactive substance synthesized and se- creted by the pineal gland and other cells [6]. The research on melatonin, which has focused on functions including regulation of biological rhythm, stabilization of gonadal function, anti-inflammatory, analgesic, and anti-oxidant ac- tivity, and immune regulation, has made great progress [7]. Melatonin’s function of regulating biological rhythms can reduce tumor risk and ameliorate the quality of life of can- cer patients [8]. It has been shown that melatonin can effec- tively alleviate breast cancer, prostate cancer, colon cancer,
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and other tumors; melatonin can also enhance the efficacy of traditional chemotherapy drugs, reduce their adverse re- actions, and prolong the life of patients [9,10]. The result of a recent study indicates that melatonin can also antagonize the effect of chemical poison-induced hepatocellular carci- noma [11]. Although the underlying molecular mechanism of the anti-cancer effect of melatonin in ACC is largely un- known, various studies have reported that the anti-tumor ability of melatonin may be mediated by a variety of mech- anisms, including antioxidant activation, inhibition of mi- gration and induction of tumor apoptosis, and reduction of endoplasmic reticulum stress [12,13]. A recent study con- firmed the treatment effect of melatonin in ACC patients [14].
The mitogen-activated protein kinase (MAPK) path- way is a key signal transduction network in eukaryotes and plays an important role in cell survival and prolifer- ation [15]. The MAPK family is divided into three cate- gories: p38 MAPK, c-jun N-terminal kinase (JNK), and ex- tracellular signal-regulated kinase 1/2 (ERK1/2) [16]. Re- cently, a study has shown that p38 MAPK and JNK sub- strates are involved in cancer cell growth and apoptosis [17], and are regulated by melatonin [18]. Another study has shown that melatonin reduces the activity of human SH-SY5 Y neuroblastoma cells through the MAPK-ERK pathway [19]. However, it is not clear whether mela- tonin affects the apoptosis of human ACC cells through the MAPK/ERK/p38/JNK signal routing. Therefore, we stud- ied the effects of melatonin on the viability and apoptosis of ACC cells, explored whether melatonin could alleviate endoplasmic reticulum stress to induce apoptosis, and the role of MAPK/ERK/p38/JNK pathway in its effects.
Materials and Methods
Cell Culture
SW-13 (Catalog number: CCL-105) and NCI-H295R (Catalog number: CC-Y1387) cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shang- hai, China). The cell lines were identified using the ap- proved DNA-based method to confirm the origin of the cell line and to check for misrecognition. The cell line was checked against the database of mistakenly identified cell lines maintained by the International Committee for Cell Line Accreditation (ICLAC). The genetic character- istics of cell lines were authenticated by short tandem re- peats (STR) profiling, and no cross-contamination was de- tected. Testing for mycoplasma indicated no contamina- tion. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) culture mediumn (C11995500BT, Mil- liporeSigma, Darmstadt, Germany) containing 10% FBS, penicillin, and streptomycin at 100 U/L each, and incu- bated in an incubator (120300, Guansen Biotechnology, Shanghai, China) at 37 ℃ with 5% CO2, and then di- gested and passaged until the cell fusion reached 80%-90%.
The logarithmic growth phase cells were taken for exper- iments. MAPK/ERK/p38/JNK pathway inhibitor nuclear factor kappa B (NF-KB)/MAPK-IN-1 obtained from Bey- otime (3.48 µM, HY-147972, Shanghai, China).
IC50 Value Detection of Melatonin
SW-13 and NCI-H295R cells in the logarithmic growth phase were inoculated in 96-well plates at a den- sity of 4000/well. The experimental grouping was con- ducted with different concentrations of melatonin (0.46875, 0.9375, 1.875 µM). Each concentration was set up with three duplicate wells, and a control grouping without mela- tonin was set up. After 24 h of drug action, 10 uL of CCK- 8 reagent was added to each well and incubated at 37 ℃ and 5% CO2 for 4 h. The optical density (OD) value at 450 nm wavelength was appraised by a microplate reader (Varioskan LUX, BioRad, Hercules, CA, USA), and half maximal inhibitory concentration (IC50) was calculated.
CCK-8 was Used to Detect Cell Activity
The cell density was reset to 1 x 104 cells/well, SW- 13 and NCI-H295R cells in the logarithmic growth phase were selected. The cells were inoculated into 96-well cul- ture plates in a 37 °℃, 5% CO2 incubator for 24 h with 100 uL per well. The cells were washed with PBS (AM9624, Posino Life Technology, Wuhan, China), and the super- natant in the culture dish was removed. Melatonin was di- luted to the corresponding concentration according to the grouping, added to each well plate, and cultured in a 5% CO2 incubator for 24 h, 48 h, and 72 h at 37 ℃, re- spectively. The cell morphology was observed by micro- scope (Olympus cx41, MICROSCOPE CENTRAL, Tokyo, Japan) at the end of the culture. Each well was augmented with 10 uL of 5 mg/mL CCK-8 (BS350A, Yisheng Biol- ogy, Nanchang, China) and incubated in a 5% CO2 incuba- tor for 1 h at 37 ℃. The OD value was gauged at the same time point at 450 nm wavelength of the microplate reader, and the measured OD value was used to analyze the effect of cell proliferation.
Wound Healing Assay
Firstly, a straight edge was used on the 3.5-cm dish and the lines were marked evenly with a marker stroke, spaced 0.5-1 cm apart for each line. SW-13 and NCI-H295R cells that were in the logarithmic growth phase were selected, and the cell density was reset to 5 x 105 cells/well. Cells were inoculated into 3.5 cm dishes and the cells were cul- tured overnight at 37 ℃ and 5% CO2 saturated humidity. When the cell density reached about 90%, the bottom of the 3.5 cm dish was covered, and the 200 uL gun head was pointed at the ruler, as far as possible perpendicular to the horizontal line scratches behind the back, with the gun head vertical, not inclined. The cells were washed 3 times with PBS, scratched cells were removed, and the cells were ob- served and photographed under the microscope. Melatonin
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was diluted to the corresponding concentration according to the grouping, added to each dish, and cultured in a 5% CO2 incubator at 37 ℃ for 72 h. At the end of the culture, cell migration was observed under a microscope. Cell mi- gration ability: migration index (%) = (initial scratch width - scratch width after healing)/initial scratch width × 100%.
Cell Invasion was Appraised by Transwell Assay
The treated SW-13 and NCI-H295R cells were washed with 3 mL PBS, digested with 0.25% trypsin (002PI, China Center for Type Culture Collection, Wuhan, China), and centrifuged at 1000 rpm for 5 min. The supernatant was removed, the cells were washed twice with PBS, and the residual serum was washed away. The cells were resus- pended in a serum-free medium and counted using a cell counting plate. The cell concentration was diluted to 3 x 105 cell/mL in a serum-free medium for later use. Ma- trigel (356234, Corning Incorporated, Corning, NY, USA) was melted a day earlier at 4 ℃, and the transwell cham- ber (3422, Corning Incorporated, Corning, NY, USA), 24- well culture plate, and the tip were precooled overnight at -20 ℃. The Matrigel was diluted with serum-free medium to a final concentration of 1 mg/mL and placed on ice. A complete medium containing 800 µL 10% FBS pre-cooled at 4 ℃ was added to 24-well plates and placed in a tran- swell chamber. 100 µL Matrigel with a final concentration of 1 mg/mL was added vertically at the bottom of the up- per chamber of the transwell chamber, and incubated at 37 ℃ for 4-5 h to make it dry into a gel. After Matrigel was dried into a gel, a 200 uL cell suspension of each grouping was added to the upper chamber of the transwell chamber, and cultured in an incubator at 37 ℃ and 5% CO2 for 24 h. Melatonin was diluted to the corresponding concentration according to the grouping with the basic medium, added to each chamber, and cultured in a 5% CO2 incubator at 37 ℃ for 72 h. The transwell was taken out, and the chamber was carefully washed with PBS, and the cells were fixed with 4% paraformaldehyde solution (P0099, Beyotime, Shang- hai, China) for 1 h. The cells were stained with 0.5% crys- tal violet staining solution, placed at room temperature for 20 min, washed with PBS, and wiped clean with a clean cotton ball. Five high-magnification fields were randomly selected from the upper, lower, left, and right sides. The number of cells in the lower chamber was counted as the number of cells penetrating the Matrigel, and the number of cells was used to represent the invasiveness of tumor cells. The number of invasive cell were counted. The experiment was repeated 3 times.
Flow Cytometry
The cell density was reset to 5 x 105 cells/well, and SW-13 and NCI-H295R cells in the logarithmic growth phase were selected. The cells were inoculated in a 6- well culture plate, and 2 mL of complete medium (L310KJ, Yuanpei Biotechnology, Shanghai, China) was added to
each well. The cells were cultured in a 5% CO2 incuba- tor at 37 ℃ for 24 h. Melatonin was diluted into the corre- sponding concentration according to the grouping, added to each well plate, and cultured in 37 ℃, 5% CO2 incubator for 72 h. Cells were collected by trypsin digestion without EDTA and washed twice with pre-cooled PBS. The cells were resuspended in 100 uL PBS, augmented with PI (5 uL, 401006, Beibo Biology, Shanghai, China), AV (5 µL), and incubated at 37 ℃ in the dark for 30 min. PBS was added to wash cells 2 times. The cells were resuspended in 300 µL PBS and appraised by flow cytometry (FACSVerse, BD, Franklin, NJ, USA).
Western Blot
The cell density was reset to 5 x 105 cells/well, and SW-13 and NCI-H295R cells in the logarithmic growth phase were selected. The cells were inoculated in 6 cm dishes, and a 3 mL complete medium was added to each dish. Before culturing cells in a 37 ℃, 5% CO2 incuba- tor for 72 h, melatonin was diluted to concentrations cor- responding to each group and added to each well plate. The old culture medium was discarded, and cells were digested with the radioimmunoprecipitation assay (RIPA) buffer (P00138, Beyotime, Shanghai, China), including 1% phenylmethanesulfonyl fluoride (PMSF) lysate and col- lected by low-speed centrifugation to extract total cell pro- tein. Protein quantification was performed using the Brad- ford method (BL521A, Biosharp, Beijing, China). The samples were boiled for 5 min, cooled on ice, and cen- trifuged for 30 s. The supernatant was subjected to poly- acrylamide gel electrophoresis (T1010, solarbio, Beijing, China) with a voltage of 100 V for 1 h. Polyvinylidene fluo- ride (PVDF) membranes (HATF00010, Millipore, Burling- ton, MA, USA) were sealed with 5% skim milk at room temperature for 1 h, and incubated overnight at 4 ℃ with primary antibodies: ERK5 (ab40809), JNK (ab307802), ERK1 (ab109282), p38 (ab170099), and glucose-regulated protein 78 (GRP78) (ab213258). Antibodies were obtained from Abcam (1:1000, Shanghai, China). After washing the membrane twice with TBST, the membrane was incu- bated with fluorescein-labeled secondary antibody (9009- 99-0, 1:2000, Abcam, Shanghai, China) at room tempera- ture for 1 h. After washing the membrane three times, the ECL chromogenic agent (ECL-0011, Dingguo Changsheng Biotechnology, Beijing, China) was exposed and imaged with a scanner (EVOSTM M7000, Thermo Fisher, Waltham, MA, USA). The gray value of each band was analyzed us- ing ImageJ software (v.1.37, NIH, Bethesda, MD, USA).
Establishment of Tumor Model in Nude Mice
Specific Pathogen Free (SPF) grade animals of 18-20 g body mass (n = 10, 6-week-old wild-type) were purchased from Sibeifu Biotechnology Co., Ltd. (Beijing, China). Mice were assigned to 2 groups: SW-13 (n = 5, control grouping) and SW-13 + Melatonin (n = 5, drug grouping).
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B
A
Proliferation(% of control)
150
0 μΜ
0.46875 µM
100
0.9375 µM
* **
**
**
**
**
1.875 μΜ
50
3.75 μΜ
7.5 µM
15 µM
0
30 μΜ
SW-13
NCI-H295R
SW-13
NCI-H295R
CK
CK
Proliferation(% of control)
150
15 μΜ
Proliferation(% of control)
150
15 µM
30 μΜ
30 μΜ
100
T
100
Z
*
T
**
**
**
**
**
**
**
**
**
50
50
0
0
24h
48h
72h
24h
48h
72h
The person performing the injection used the left index fin- ger and thumb to pinch the back of the mouse with the ab- domen facing them. The left index finger was used to fix the right forelimb, exposing the right armpit of the mouse, and the needle was inserted into the armpit. Each mouse was injected with 1 × 107 cells with an injection volume of 100 uL/mouse. For the SW-13 + Melatonin group, Mela- tonin (100 mg/kg/d) was injected intraperitoneally every day when the tumor size reached 0.2 x 0.2 cm2; the SW-13 group was given an equal amount of saline by gavage. De- tection during modeling: The body weight and tumor size of mice were measured every 5 days for 15 days. After 15 days of modeling, the mice were euthanized. After anesthe- sia (0.3% sodium pentobarbital solution, 40 mg/kg), bled to death, photographed, and the tumor was frozen in liquid nitrogen and stored at -80 ℃. All experimental protocols
of this study were approved by Zhuoqiang Biotechnology, Co., Ltd. ethics committee (No: ZQZA-2023-021).
Statistical Analysis
Statistical analysis was performed using SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, USA). Nor- mally distributed variables were expressed as mean ± stan- dard deviation (ī ± s). T-tests were used for comparisons between two groups, and analysis of variance was used for comparisons between multiple groups. Post hoc analysis was conducted using the Tukey test. p < 0.05 was consid- ered statistically significant.
DM
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A
SW-13
NCI-H295R
100
Melatonin 15 uM Melatonin 30 µM
CK
CK
CK
Melatonin 15 uM Melatonin 30 µM
Migration rate (%)
80
**
15uM
**
0h
**
30μΜ
60
100 pr
100 pm
100 pm
100 DAY
100 pm
100 um
40
**
72h
20
100 110
100 pm
100
100 pm
100 pm
100 pm
0
T
SW-13
NCI-H295R
T
B
CK
Melatonin 15 µM
Melatonin 30 µM
600
CK
SW-13
Invasion cell number
T
15uM
400
30μΜ
200 um
200 pm
#
200 um
CK
Melatonin 15 µM
Melatonin 30 µM
**
NCI-H295R
200
**
**
**
200 -şim
200 um
200 pim
0
T
T
SW-13
NCI-H295R
C
CK
Melatonin 15 µM
Melatonin 30 µM
104
10
104
Q1
Q2
01
Q2
Q1
2.77
6.32
3.07
23.5
0.040
Q2
18.4
10%
10ª
10°
SW-13
FL3-H :: PI
₹ 4 FL3-H :: PI
FL3-H :: PI
40
CK
10ª
10
10
Apoptosis rate (%)
**
10
101
10
30
15uM
Q4
03
04
03
Q4
**
30µM
90.4
0.54
70.5
10ª
2.94
03
65.4
**
10
13.2
100
101
102
103
10
10ª
10’
102
10ª
10
10
10ª
10’
102
10ª
104
FLI-H : AV-ITC
FL1-H :: AV-FITO
FL1-H : AV-FITO
CK
Melatonin 15 µM
Melatonin 30 μM
20
**
104
104
Q1
104
Q1
02
02
Q1
0.000
0.060
9.86
0.060
Q2
4.11
15.2
NCI-H295R
10%
10°
10°
10
FL3-H :: PI
E 1 FL3-H :: PI
& 2
102
10
10
2
101
10
0
10
T
Q4
q3
03
04
G3
SW-13
NCI-H295R
93.8
10°
1.99
04
83.7
6.42
10º
101
102
103
104
10ª
10ª
10’
102
10ª
104
10ª
76.2
8.54
101
102
10ª
104
FLI-H : AV-ITC
FL1-H :: AV-FITG
10ª
FL1-H : AV-FITG
Results
Melatonin Restrained the Proliferation Rate of ACC Cells
Cell proliferation rate was substantially attenuated in the melatonin groups, compared with that of the 0 uM group. Cell proliferation rate decreased with increased melatonin concentration, with the lowest rate observed in the 30 uM group. The IC50 value of SW-13 cells was 103.114 µM, and the IC50 value of NCI-H295R cells was 348.781 µM, which was authenticated via CCK-8 assay. Subsequently, 15 uM and 30 µM concentrations were se- lected for experiments (p < 0.05, Fig. 1A). In contrast with the Control Check (CK) group, the cell proliferation rate of the melatonin 15 uM and 30 uM groups decreased substan- tially, and the cell proliferation rate was the lowest when
the melatonin concentration was 30 uM and the treatment time was 72 h, which was validated via CCK-8 assay (p < 0.05, Fig. 1B). Subsequently, a Melatonin drug concentra- tion of 30 µM and a treatment time of 72 h were selected for SW-13 cells.
Melatonin Restrained Metastasis of ACC Cells and Induced Apoptosis
Compared with the CK group, migration rates of SW- 13 and NCI-H295R cells were substantially reduced after treatment with melatonin at 15 uM and 30 uM for 72 h, which was validated via the wound healing experiment (p < 0.01, Fig. 2A). Compared with the CK group, the invasion ability of SW-13 and NCI-H295R cells was substantially reduced after treatment with melatonin at 15 uM and 30 uM for 72 h, which was validated via transwell invasion
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SW-13
NCI-H295R
ERK5
0.25
CK
2.0
15uM
T
CK
0.20
T
JNK
ERK5/GAPDH
30μΜ
JNK/GAPDH
1.5
T
15uM
**
ERK1
0.15
T
30μΜ
P38
1.0.
0.10
GRP78
0.05
0.5
GAPDH
CK
Melatonin 15 µM
Melatonin 30 µM
CK
Melatonin 15 µM
Melatonin 30 µM
0.00
0.0
SW-13
NCI-H295R
SW-13
NCI-H295R
1.5
CK
2.5
1.0
CK
15uM
CK
ERK1/GAPDH
0.8
T
15UM
30μΜ
P38/GAPDH
2.0
15uM
GRP78/GAPDH
1.0
30μΜ
30μΜ
*
T
0.6
*
1.5
0.4-
0.5-
1.0
0.5
0.2-
0.0
0.0
SW-13
T
0.0
NCI-H295R
SW-13
NCI-H295R
SW-13
NCI-H295R
assay (p < 0.01, Fig. 2B). Flow cytometry confirmed that, compared with the CK group, the apoptosis rate of SW-13 and NCI-H295R cells was substantially increased in 15 uM and 30 µM melatonin concentration groups after 72 h (p < 0.01, Fig. 2C).
Melatonin Reduced Endoplasmic Reticulum Stress and MAPK/ERK/p38/JNK Signal Routing in an in Vitro Model
Compared with the CK group, the expression levels of ERK/p38/JNK pathway proteins (ERK5, JNK, ERK1, p38) and endoplasmic reticulum stress-related proteins (GRP78) in SW-13 and NCI-H295R cells were substantially reduced after treatment with melatonin at 15 uM and 30 µM for 72 h, which was validated via western blot assay (p < 0.0001, Fig. 3), suggesting melatonin could reduce endoplasmic reticulum stress and MAPK/ERK/p38/JNK signal routing in an in vitro model.
Melatonin Increased Apoptosis of ACC Cells by Reducing Endoplasmic Reticulum Stress and MAPK/ERK/p38/JNK Signal Routing in Vitro
SW-13 cells were treated with the MAPK/ERK/p38/JNK pathway inhibitor NF-KB/MAPK- IN-1 (3.48 uM, HY-147972, Biotech Biotech, Wuhan, China). In contrast with the CK group, the melatonin- treated groups displayed substantially reduced cell proliferation, and the cell proliferation rate was further reduced after NF-KB/MAPK-IN-1 treatment (p < 0.01, Fig. 4A). Compared with the CK group, cell migration rate and invasive cell number were substantially abated after treatment with Melatonin and the inhibitor (p < 0.01, Fig. 4B,C). Melatonin significantly reduced the cell proliferation rate and increased the apoptosis rate of SW-13 cells compared with the CK group, and these effects were further enhanced after inhibitor treatment (p < 0.001, Fig. 4D). Western blot results showed that compared with the CK group, the expression levels of JNK, p38, and GRP78 were significantly reduced after Melatonin treatment, and these reductions were enhanced after inhibitor treatment (p < 0.001, Fig. 4E).
DM
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A
B
CK
Melatonin 30 µM
NF-KB/MAPK-IN-1
80-
CK
Migration rate (%)
150
Melatonin 30 µM
60-
Proliferation(% of control)
NF-KB/MAPK-IN-1
0 h
**
40-
100-
100 gr
**
20
-
50-
72 h
0
CK
Melatonin 30uM
NF-KB/MAPK-IN-1
0
100 pro
24h
48h
72h
D
C
Invasion cell number
500-
104
CK
104
Melatonin 30 μM
104
NF-KB/MAPK-IN-1
Q1
Q2
01
CK
Melatonin 30 µM
NF-KB/MAPK-IN-1
400
0
2.52
0
02
Q2
11.8
0
15.5
103
103
103
300
FL3-H :: PI
2 1 FL3-H :: PI
” FL3-H :: PI
200
102
102
102
**
*
100-
101
101
10
200 jh:
200 Km -
200 um
Q4
Q3
3.67
Q4
70.4
Q3
04
0
17.9
51.5
03
22.9
CK
Melatonin 30uM
NF-KB/MAPK-IN-1
10º
10º
101
102
103
10
10°
10º
101
102
103
104
10º
10º
101
102
103
FL1-H : AV-FITC
104
FL1-H :: AV-FITC
FL1-H : AV-FITC
50
Apoptosis rate (%)
40
E
0.5-
0.4
0.5-
T
30
JNK
JNK/GAPDH
0.4-
GRP78/GAPDH
0.3-
0.4
P38/GAPDH
20
GRP78
0.3
0.3
0.2-
10
P38
0.2-
0.2-
0.1
0.1-
0.1
0
GAPDH
CK
Melatonin 30uM
NF-KB/MAPK-IN-1
CK
Melatonin 30 µM
NF-KB/MAPK-IN-1
0.0
CK
Melatonin 30uM
NF-KB/MAPK-IN-1
0.0
CK
0.0
Melatonin 30uM
NF-KB/MAPK-IN-1
CK
Melatonin 30uM
NF-KB/MAPK-IN-1
Melatonin Increased Apoptosis of Adrenocortical Carcinoma Cells by Reducing Endoplasmic Reticulum Stress
To evaluate the mechanism of melatonin-induced apoptosis of ACCs in vivo, a subcuta neous tumor model in nude mice (SW-13) was constructed. The comparison of body weight of nude mice showed that there was no sig- nificant difference in body weight between groups. The comparison of tumor mass in nude mice showed that tumor mass in the melatonin treatment group was substantially re- duced compared with the saline group (p < 0.05, Fig. 5A). Compared with the saline group, expression of p38 and JNK was significantly reduced, and expression of C/EBP homol- ogous protein (CHOP) increased significantly after Mela- tonin treatment, which was authenticated via western blot (p < 0.01, Fig. 5B).
Discussion
At present, ACC is mainly treated by surgery. The drug treatment of ACC employs an EDP-M (etoposide, adriamycin, cisplatin combined with mitotane) regimen, but the therapeutic effect of this regimen is not satisfactory. The median progression-free survival is only 5 months, and the overall survival rate is only 14.8 months. Most patients cannot tolerate its toxicity and side effects [20]. Tradi- tional Chinese medicine has attracted the attention of schol- ars both domestically and internationally due to its natural and minimal toxic side effects. With continued research, the anti-tumor effects of traditional Chinese medicine are re- ceiving increasing attention, and some drugs have been ap- proved by USA FDA for clinical phase II trials [21]. Mela- tonin is a kind of neuroendocrine hormone, which has at- tracted more and more attention because of its wide distri- bution, low toxicity, and good histocompatibility [22]. In the current study, we confirmed the anti-tumor effects of melatonin in ACC through basic experimental studies.
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A
SW-13
SW-13
SW-13+Melatonin
SW-13+Melatonin
**
25-
600-
1.5
Weight (g)
Tumor volume (mm3)
Tumor weight (g)
20-
*
400-
1.0.
15-
**
10-
200-
0.5
5
0
0.0
7d
13d
18d
23d
0
7d
13d
18d
23d
SW-13+Melatonin’
time (days)
time (days)
SW-13
B
0.8
0.6
**
1.0
CHOP
CHOP/GAPDH
T
0.6
JNK/GAPDH
0.8
P38/GAPDH
0.4
JNK
0.6
0.4
**
**
P38
T
0.4
T
0.2
GAPDH
0.2
0.2
SW-13
SW-13+Melatonin
0.0
0.0
0.0
SW-13
SW-13+Melatonin
SW-13
SW-13+Melatonin
SW-13
SW-13+Melatonin
At present, it is believed that the mechanisms of mela- tonin mainly involve anti-tumor cell proliferation, induc- tion of tumor cell apoptosis, regulation of immune function, and inhibition of tumor cell invasion and metastasis [23]. A previous study has shown that melatonin can down-regulate protein kinase B (Akt) and murine doubleminute 2 (MDM2) to promote apoptosis of human gastric cancer cells AGS and MGC803 [24]. Wang et al. [25] have shown that mela- tonin treatment substantially reduced tumor size in a subcu- taneous oral cancer xenograft model without obvious sys- temic side effects, substantially enhanced apoptosis and fer- roptosis levels in tumor tissues, and abated autophagy lev- els. Melatonin-assisted cisplatin alleviates the proliferation of bladder cancer cells by abating PrP-regulated cell stress and cell proliferation signals [26]. Glyphosate and hard wa- ter synergistically promote proximal renal tubular epithelial cell aging through PINK1 Parkin-mediated mitochondrial autophagy, whereas melatonin exerts renal protective ef- fects by regulating mitochondrial autophagy [27]. A previ- ous study has also preliminarily confirmed the therapeutic effect of melatonin in ACC [14]. The present study con-
firmed, through cytology and animal model studies, that, compared to the CK group, melatonin reduced the prolifer- ation, migration, and invasion rates of ACC cells, and sub- stantially induced cell apoptosis.
The expression level of GRP78 is relatively low in mature organs, but it is elevated in tumor tissues. The tumor microenvironment has characteristics such as low sugar, hypoxia, and acidosis, which can cause the aggrega- tion of non-glycosylated and non-folding proteins in the en- doplasmic reticulum, thereby initiating endoplasmic retic- ulum stress [28]. In the present study, melatonin interven- tion reduced the level of endoplasmic reticulum stress pro- tein GRP78. The MAPK family is an important intracel- lular signal routing. Under the influence of different stim- uli, the MAPK family regulates various physiological func- tions such as cell proliferation, growth, death, and migra- tion. The generation of these functions is achieved through processes such as activation and inactivation of the MAPK family within cells [29]. The present study evaluated the role of MAPK/ERK/p38/JNK signal routing in melatonin- induced apoptosis of ACC cells and inhibition of endo-
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plasmic reticulum stress. The results showed that mela- tonin intervention could reduce the protein expression re- lated to MAPK/ERK/p38/JNK signal routing (ERK5, JNK, ERK1, p38). MAPK/ERK/p38/JNK pathway inhibitor NF- KB/MAPK-IN-1 (3.48 µM) intervention enhanced the in- hibitory effect of melatonin on ACC cell activity and in- duced cell apoptosis. It was further confirmed that mela- tonin induces ACC cell apoptosis by reducing endoplasmic reticulum stress, and MAPK/ERK/p38/JNK signal routing was associated with this effects. However, further evalua- tion of these mechanisms is needed.
Conclusion
In summary, the results of this study confirm that melatonin induces ACC cell apoptosis and alleviates en- doplasmic reticulum stress, and this effect was related to the inhibition of the MAPK/ERK/p38/JNK. It provides an experimental basis for the use of melatonin in the clinical treatment of ACC.
Availability of Data and Materials
The data generated or analyzed during this study are included in this published article or obtained from the cor- responding author on reasonable request.
Author Contributions
QSL and SS contributed to the concept and designed the research study. SS performed the research. PCH pro- vided help and advice on the research study. QSL analyzed the data. HYY, ZXH, DCL wrote the manuscript and made substantial contributions to the conception and design, ac- quisition of data and analysis of data. ZJL helped analysis with constructive discussion. All authors contributed to the important editorial changes in the manuscript. All authors agreed to be accountable for all aspects of the work in en- suring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and re- solved. All authors read and approved the final manuscript.
Ethics Approval and Consent to Participate
All experimental protocols of this study were ap- proved by Zhuoqiang Biotechnology, Co., Ltd. ethics com- mittee (No: ZQZA-2023-021).
Acknowledgment
Not applicable.
Funding
This study was supported by the National Natural Sci- ence Foundation of China (grant number: 81060220).
Conflict of Interest
The authors declare no conflict of interest.
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