Effects of Birinapant on Proliferation and Invasion of MGC-803 Gastric Cancer Cells and Mechanism Underlying These Effects
D. L. Zhu and L. Y. Shuai
Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 171, No. 1, pp. 73-79, January, 2021 Original article submitted April 21, 2020

We studied the effects of birinapant, a mimetic of the second mitochondria­derived activa- tor of caspase (SMAC), on invasion and proliferation of MGC-803 gastric cancer cells and the molecular mechanisms underlying these processes. The expression of cellular inhibitor of apoptosis 1 (cIAP1) and TNF receptor-associated factor 3 (TRAF3) in gastric cancer cell line MGC­803 and normal gastric mucosa GES­1 cells were analyzed by Western blotting and cell immunofluorescence assay. After pretreatment of MGC­803 cells with birinapant, a Transwell invasion assay was used to evaluate the cell invasion ability. MGC­803 cells were implanted under the skin of BALB/c nude mice. The tumors were removed 10 days later and its size was measured. Protein expression of proliferating cell nuclear antigen (PCNA) in the subcutaneous tumors was analyzed by immunohistochemical method. In addition, the expression of cIAP1, TRAF3, pNF­κB, and NF­κB in control and birinapant­treated cells was compared by Western blotting and the rate of cell apoptosis was evaluated by flow cytometry. In untreated MGC­803 gastric cancer cells, the expression of cIAP1 was higher and the ex- pression of TRAF3 was lower than in normal gastric mucosa cell line GES­1. Pretreatment with birinapant inhibited the invasion and proliferation of MGC­803 cells and promoted cell apoptosis. Birinapant also promoted the expression of TRAF3 and inhibited the expression of cIAP1 and pNF­κB in MGC­803 cells. Thus, birinapant inhibited the expression of cIAP1, prevented degradation of TRAF3, and suppressed invasion and proliferation of MGC-803 cells by promoting cell apoptosis.

Key Words: gastric cancer; SMAC mimetic; birinapant; invasion; proliferation

Gastric cancer is one of the most common malignant tumors of the gastrointestinal tract [3]. Due to the lack of specific markers, specific symptoms, and signs at the early stage, the detection rate of early gastric can- cer is low [8]. For advanced gastric cancer, surgical resection of the tumor is the most common method of

Department of General Surgery of Jiangjin Central Hospital of Chong- qing, Chongqing, China. Address for correspondence: shuaileiyu- [email protected]. L. Y. Shuai
treatment that can also be combined with radiotherapy and chemotherapy [2]. However, the curative effect is poor, and the overall survival remains low [2]. There- fore, further studies of the molecular mechanism of gastric cancer occurrence and development are ex- tremely important for its early diagnosis and treatment. Inhibitors of apoptosis proteins (IAPs) regulate diverse cell functions, including mitosis, migration, apoptosis, and signal transduction events, that promote inflammation [10]. cIAP1, one of the main members

0007-4888/21/1711-0056 © 2021 Springer Science+Business Media, LLC

of the IAP family, regulates cell apoptosis [1]. TNF receptor-associated factor 3 (TRAF3) plays an im- portant role in immune diseases and tumor growth [14]. Second mitochondria-derived activator of cas- pases (SMAC) exists in mitochondria and regulates cell apoptosis [4]. Studies have shown that SMAC and SMAC mimetics can promote tumor cell apoptosis and inhibit malignant biological behavior of tumor cells by activating cIAP1 [6]. SMAC mimetic birinapant po- tentiates bortezomib antimyeloma activity in vitro and in vivo by inhibiting the cIAP1/p52 signaling pathway [15]. Birinapant enhances PD-1-mediated antitumor immunity and improves the efficiency of combination therapy through co-inhibition of the immune check- point by promoting the production of TNF [5]. In addition, SMAC mimetic birinapant attenuates LPS- induced liver injury by inhibiting TRAF3 degradation in Kupffer cells [7]. However, the effect of birinapant, a typical mimetic of SMAC, on the development of gastric cancer and the molecular mechanism underly- ing this effect are still unknown.
The objective of the present study was to explore the effect of SMAC mimetic birinapant on prolifera- tion and invasion of MGC-803 human gastric cancer cells both in vitro and in vivo and the molecular mech- anism underlying this effect. The results can provide experimental basis for further study of the mechanism of gastric cancer and exploration of new therapeutic targets for gastric cancer.

MATERIALS AND METHODS
Cell culture. The human gastric cancer cell line MGC- 803 and the normal gastric mucosa cell line GES-1 were obtained from the Chinese Academy of Science (Shanghai). These cells were cultured in DMEM sup- plemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°C, 95% humidity, and 5% CO2.
Subcutaneous tumor model in mice. Male
BALB/c nude mice (age 7­8 weeks) were obtained from the Animal Experimental Center of the Chongq- ing Medical University. This study was approved by the Ethics Committee of Jiangjin Central Hospital of Chongqing and performed in accordance with Asso- ciation for Assessment and Accreditation of Labora- tory Animal Care guidelines and Directive 2010/63/ EU (On the Protection of Animals used for Scientific Purposes; September 22, 2010).
The mice were divided into the control and treat- ment groups (5 mice per group). In both groups, the tumor was modelled by subcutaneous injection of 107 MGC-803 cells. The mice in the treatment group were injected intraperitoneally with 20 mg/kg birina- pant (two 5­day courses of daily injections); controls
were injected with the same volume of normal saline at the same time points. Subcutaneous tumors were removed 10 days later for the follow­up study.
Western blotting. The total protein in each group of cells was extracted by using the KeyGEN total protein extraction kit. Then, the protein con- centration was measured using a BCA protein as- say kit (Beyotime). The protein solution was mixed 4:1 (v/v) with SDS­PAGE Protein Loading Buffer 5× (Boster) and boiled in water for 10 min. Then, proteins were first resolved by SDS­PAAG electrophoresis and transferred to PVDF membranes that were blocked with 5% BSA for 1 h at room temperature. The mem- branes were incubated overnight at 4°C with primary antibodies against cIAP1 (1:1000; ab196592; Ab- cam), TRAF3 (1:1000; ab263026; Abcam), p­NK­κB (1:1000; ab86299; Abcam), NK­κB (1:1000; ab16502;
Abcam) and β­actin (1:1000; #3700; Cell Signaling Technology, Inc.) and then with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000; #7074; Cell Signaling Tech- nology, Inc.) at 37°C for 1 h followed by incubation with enhanced chemiluminescence reagent (Pierce) at room temperature for 5 sec. Then, the membranes were analyzed and scanned using Quantity One soft- ware (Bio­Rad).
Quantitative real-time PCR with reverse tran- scription (RT-qPCR). Total RNA of MGC-803 and GES­1 cells was extracted using TRI reagent (Sigma) according to manufacturer’s instructions and reverse transcription to cDNA was performed using Prime- Script RT Reagent Kit with gDNA Eraser (Perfect Real Time) (RR047A, Takara). Finally, RT­qPCR was carried out. The gene expression levels were norma­ lized to those of GAPDH. The primer sequences are presented in Table 1.
Cell immunofluorescence. MGC-803 and GES- 1 cells (103 each) were inoculated into 24­well plates containing coverslips and 500 µl of complete culture medium was added to each well. In 24 h, the cells were washed 3 times with precooled PBS and fixed with 4% paraformaldehyde at 37°C for 10 min; the cell membranes were permeabilized with 0.2% Triton for 10 min. The coverslips were washed with PBS 3 times and then blocked with goat serum for 1 h. cIAP1 antibody (dilution 1:500) was added and incu- bated overnight at 4°C. The next day, the coverslips were washed with PBS (3×5 min) and then incubated with the corresponding fluorescent secondary antibody (ZSGB Bio). After 1-h incubation at room tempera- ture, the coverslips were washed 3 times with PBS, an appropriate amount of DAPI was added to each cov- erslip for 3 min, and the coverslips were sealed with an anti­fluorescence quencher. Then, cIAP1 expression was evaluated under an inverted microscope.

Immunohistochemistry. Immunohistochemi- cal analysis was performed as previously described [13]. Staining of subcutaneous tumor tissues (n=5) involved a series of steps, such as fixing, embedding, slicing, dewaxing, rehydrating, and antigen retrieval. The slides were blocked with 5% normal goat serum (Beyotime) at room temperature for 1 h. Then, the slides were incubated overnight with PCNA antibody (1:100; ab92552; Abcam) at 4°C. On the next day, the slides were incubated with secondary antibody for 1 h. Then, after staining with DAB solution (Boster) and hematoxylin, the sections were dehydrated and embedded with neutral gum. Finally, the expression of PCNA was evaluated under a light microscope at
×200.
Cell invasion assays. MGC­803 cells were resus- pended in culture medium with 8% FCS and applied to the upper Transwell chamber containing Matrigel (BD Biosciences); 800 μl culture medium with 15% FCS was added to the lower chamber. The cells were incu- bated for 24 h. After fixation with 4% paraformalde- hyde and staining with a 0.1% crystal violet solution, the cells on the upper surface of the filter membrane were removed. Then, the cells were counted under a light microscope.
Cell apoptosis assays. Cell suspension (106 cells in 500 μl medium) was incubated with 5 μl annexin V (Boster) and 5 μl PI in the dark at room temperature for 10 min. The rate of apoptosis (early+late stages) was determined by flow cytometry within 1 h.
Statistical analysis. Statistical analyses were performed using SPSS Statistics 17.0 software (IBM, Inc.). The Student’s t test was used for the comparison of parameters between two groups. One­way ANOVA was used to compare the parameters among more than two groups. The differences were significant at p<0.05.

RESULTS
The expression of cIAP1 and TRAF3 in MGC-803 cells. In gastric cancer cell line MGC-803, the expres- sion of cIAP1 protein and mRNA was significantly higher, while the expression of TRAF3 protein and mRNA was significantly lower than in normal gastric mucosa GES-1 cells (Fig. 1, a, b). Cell immunofluo- rescence assay also showed that cIAP1 was highly expressed in MGC­803 cells and was expressed at low levels in GES-1 cells (Fig. 1, c). These findings sug- gest that cIAP1 is highly expressed in MGC-803 cells, while TRAF3 is expressed at low levels.
Birinapant inhibits proliferation and invasion of MGC-803 cells. MGC­803 cell cultures were di- vided into the control (untreated) and treatment (24- h incubation with 300 nmol/ml birinapant) groups. Transwell assay showed that pretreatment with biri-
napant significantly inhibited invasion of MGC­803
cells (Fig. 2, a).
In vivo experiments on mice showed that the size and weight of subcutaneous tumors in the birinapant group were significantly lower than in the control group (Fig. 2, b). Immunohistochemical analysis of the tumors showed that the expression of PCNA in the birinapant group was significantly lower than in the control group. Hence, birinapant inhibits prolif- eration of MGC-803 cells (Fig. 2, c). Thus, treatment with birinapant inhibited proliferation and invasion of MGC-803 cells.
Birinapant inhibits cIAP1 expression and pro- motes degradation of TRAF3. Studies have reported that SMAC is directly involved in the regulation of cIAP1 expression and that cIAP1 can regulate cell apoptosis by affecting degradation of TRAF3 [7]. To further study the molecular mechanism by which birinapant inhibits the proliferation and invasion of MGC­803 cells, Western blotting was used to detect the protein expression of cIAP1, TRAF3, pNF­κB, and NF­κB in the two groups of cells. It was found that pretreatment with birinapant significantly inhibited the expression of cIAP1 and pNF­κB and stimulated the expression of TRAF3 (Fig. 3, a). Flow cytometry showed that pretreatment with birinapant promoted apoptosis of MGC-803 cells (Fig. 3, b). The above results indicated that SMAC mimetic birinapant inhi- bited the expression of cIAP1, prevented degradation of TRAF3, and inhibited the invasion and proliferation of MGC-803 cells by promoting cell apoptosis.
Gastric cancer is the fifth most common cancer, the second most common type of cancer, and a lead- ing cause of cancer­related mortality worldwide [3,8]. Although the incidence of gastric cancer has declined in recent decades, the total number of cases is increas- ing, mainly due to the aging of the population [2,8]. In terms of diagnosis, the lack of specific detection factors and the lack of specific symptoms and signs at the early stages lead to low detection rate of early gastric cancer. In terms of treatment, early gastric can- cer has a good therapeutic outcome and can even be completely cured [8]. Therefore, further research on the pathogenesis and development mechanism of gas- tric cancer is of great significance in developing new therapeutic methods and targets and improving the survival rate of patients.
Recent studies have shown that the occurrence and development of gastric cancer are closely related to multiple genes and multiple pathways. Cancer­asso- ciated fibroblasts in the tumor microenvironment pro- mote the progression of gastric cancer through IL-6/ JAK2/STAT3 signaling [13]. CDGSH iron sulfur do- main 2 (CISD2) is localized in the outer mitochondrial membrane and mediates mitochondrial integrity and

GES-1 MGC-803

cIAP1

TRAF3

-аctin

cIAP1 TRAF3

cIAP1 DAPI Merged images

GES-1

MGC-803

Fig. 1. The expression of cIAP1 and TRAF3 proteins in normal (GES-1) and gastric cancer (MGC-803) cells. a) Western blotting,
b) RT-qPCR, c) immunofluorescence. *p<0.05, **p<0.01 in comparison with GES-1 cells.

lifespan in mammals. Overexpression of CISD2 has prognostic value in human gastric cancer and promotes gastric cancer cell proliferation and tumorigenesis via the Akt signaling pathway [11]. Stress­responsive mul- tifunctional protein RLIP76 is usually overexpressed in malignant carcinomas. In gastric cancer tissues, RLIP76 was overexpressed and knockdown of RLIP76 in MGC­ 803 cells by shRNA-RLIP76 increased cell apoptosis through the Akt/mTOR signaling pathway [12].
SMAC is a new type of mitochondrial protein and an inhibitor of IAPs [4]. It can induce degrada- tion of TRAF3 and enhance activity of caspase-3, and promote cell apoptosis through interaction with IAPs [4,6]. With further understanding of the structure and
function of SMAC, SMAC mimetic came into being. A SMAC mimetic is a small molecule IAP inhibitory protein that mimics the antagonist SMAC of endog- enous IAPs, which can well reflect the biological func- tion of SMAC to a certain extent [6]. SMAC mimetics interact with IAPs to promote apoptosis and success- fully inhibit the development of various tumors, such as liver cancer, head and neck malignant tumors, and breast cancer [9,15].
In this study, we found that the expression of cIAP1 was high and the expression TRAF3 was low in MGC­803 cells in comparison with normal gastric mucosa GES­1 cells. Pretreatment with birinapant in- hibited the invasion and proliferation of MGC-803

Control Birinapant
Cell number
80

MGC-803
60

40

20

0
Control Birinapant

Control Birinapant
g
1.5

1.0

0.5

0
Control Birinapant

Control Birinapant
PCNA -cells
+
200

150

PCNA
100

50

0
Control Birinapant
Fig. 2. The effect of birinapant on proliferation and invasion of MGC-803 cells. a) Invasion ability of MGC-803 cells (Transwell assay,
×200), b) the size and weight of subcutaneous tumors in control and birinapant-treated mice, c) expression of PCNA in subcutane- ous tumors (immunohistochemical analysis, ×200). *p<0.05 in comparison with the control.

670/30 [561]-PI
3
10
cIAP1
Control Birinapant

4
10
R2-Q1 R2-Q2

670/30 [561]-PI
3
10
4
10
R2-Q1 R2-Q2

1
10
1
10
TRAF3 p-NF- B
P2+P3=1.35%
P2+P3=17.41%

2
10
2
10
NF- B

β-actin

0
10
R2-Q4

R2-Q3

0
10
R2-Q4

R2-Q3

0 1 2 3 4
10 10 10 10 10
530/40 [488]-Annexin V FITC
10 10 10 10 10
0 1 2 3 4
530/40 [488]-Annexin V FITC

Fig. 3. The molecular mechanism of the effect of birinapant on MGC-803 cells. a) Expression of cIAP1, TRAF3, pNF-κB, and NF-κB proteins (Western blotting), b) apoptosis of MGC-803 cells in the control and after treatment with birinapant (flow cytometry).

cells and promoted cell apoptosis. In addition, biri- napant also promoted the expression of TRAF3 in MGC-803 cells and inhibited the expression of cIAP1 and pNF­κB. Therefore, we could see that birinapant inhibited the expression of cIAP1, inhibited the deg- radation of TRAF3, and further inhibited the invasion and proliferation of MGC-803 cells by promoting cell apoptosis. The results of this study provide a theoreti- cal basis for the clinical treatment of gastric cancer. However, there are still some limitations to this study. For example, the research on the mechanism was not deep enough. In addition to MGC-803, gastric cancer cell lines also include SGC-790, MKN45, and AGS cells. Whether birinapant plays the same role in the de- velopment of these gastric cancer cells is still unclear.
Authors declare no conflict of interest.

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