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Cited by 261Cancer, Stress, Anesthesia, and Immune ResponseReceptor Mechanisms and SignalingGastric Cancer Management and Outcomes

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Chronic stress promotes gastric cancer progression and metastasis: an essential role for ADRB2

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Zhang, Xuan, Zhang, Yi, He, Zhongyuan, Yin, Kai, Li, Bowen, Zhang, Lu, Xu, Zekuan·2019·Cell Death & Disease
DOI 10.1038/s41419-019-2030-2PMC PMC6797812
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Abstract

An increasing number of studies indicate that adrenergic signalling plays a fundamental role in chronic stress-induced tumour progression and metastasis. However, its function in gastric cancer (GC) and its potential mechanisms remain unknown. The expression levels of β-adrenergic receptor (ADRB) in GC cell lines were examined by using real-time polymerase chain reaction (RT-PCR) and western blotting. The effects of β2 adrenergic receptor (ADRB2) activation and blockade were investigated in vitro in GC cells by using proliferation, migration, invasion, cell cycle and apoptosis assays. Chronic restraint stress (CRS) increased the plasma levels of catecholamines and cortisol and also induced progression and metastasis of GC in vivo. Furthermore, immunohistochemical staining and a TUNEL assay were employed to observe the regulation of cell viability in vivo. The expression levels of ADRB2 in 100 human GC samples were measured by RT-PCR and immunohistochemistry. The stress hormones epinephrine and norepinephrine significantly accelerated GC cell proliferation, invasion and viability in culture, as well as tumour growth in vivo. These effects were reversed by the ADRB antagonists propranolol and ICI118,551 (an ADRB2-specific antagonist). Moreover, the selective ADRB1 antagonist atenolol had almost no effect on tumour cell proliferation and invasion in vitro and in vivo. ADRB2 antagonists suppressed proliferation, invasion and metastasis by inhibiting the ERK1/2-JNK-MAPK pathway and transcription factors, such as NF-κB, AP-1, CREB and STAT3. Analysis of xenograft models using GC cells revealed that ADRB2 antagonists significantly inhibited tumour growth and metastasis, and chronic stress antagonized these inhibitory effects. In addition, chronic stress increased the expression of VEGF, MMP-2, MMP-7 and MMP-9 in transplanted tumour tissue, and catecholamine hormones enhanced the expression of metastasis-related proteins. The expression of ADRB2 was upregulated in tumour tissues and positively correlated with tumour size, histological grade, lymph node metastasis and clinical stage in human GC samples. Stress hormone-induced activation of the ADRB2 signalling pathway plays a crucial role in GC progression and metastasis. These findings indicate that ADRB2 signalling regulates GC progression and suggest β2 blockade as a novel strategy to complement existing therapies for GC.

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Fig. 1 ADRB expression in human gastric tissues and GC cells. a qRT-PCR analysis of the ADRB mRNA levels in GC cell lines and GES-1 cells. b, c Western blotting was used to analyse the expression level of ADRB protein in GES-1 cells and GC cell lines. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, or ***p < 0.001
Fig. 1 ADRB expression in human gastric tissues and GC cells. a qRT-PCR analysis of the ADRB mRNA levels in GC cell lines and GES-1 cells. b, c Western blotting was used to analyse the expression level of ADRB protein in GES-1 cells and GC cell lines. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, or ***p < 0.001CC BY · PubMed Central
Fig. 2 Effect of β-adrenergic signalling on the proliferation of GC cells in vitro. a, b Effects of non-specific ADRB agonists and blockers on HGC27 and MGC803 cell proliferative ability by CCK8 assay. c, d The effects of specific ADRB agonists and blockers on the proliferation potential of HGC27 and MGC803 cells were determined by a CCK8 assay and are presented as a growth curve. e, f Knockdown of ADRB2 expression, and CCK8 validation of various types of ADRB agonists on the proliferation of gastric cancer. g The EdU assay validated the functional role of various types of ADRB agonists and antagonists in HGC27 cell proliferation. h, i A colony-forming assay was used to detect the effects of various specific ADRB agonists and blockers on the colony-forming ability of HGC27 and MGC803 cells. Representative images are shown. j, k, l, m The efficiency of ADRB2 knockout was determined by real-time quantitative PCR and immunoblotting
Fig. 2 Effect of β-adrenergic signalling on the proliferation of GC cells in vitro. a, b Effects of non-specific ADRB agonists and blockers on HGC27 and MGC803 cell proliferative ability by CCK8 assay. c, d The effects of specific ADRB agonists and blockers on the proliferation potential of HGC27 and MGC803 cells were determined by a CCK8 assay and are presented as a growth curve. e, f Knockdown of ADRB2 expression, and CCK8 validation of various types of ADRB agonists on the proliferation of gastric cancer. g The EdU assay validated the functional role of various types of ADRB agonists and antagonists in HGC27 cell proliferation. h, i A colony-forming assay was used to detect the effects of various specific ADRB agonists and blockers on the colony-forming ability of HGC27 and MGC803 cells. Representative images are shown. j, k, l, m The efficiency of ADRB2 knockout was determined by real-time quantitative PCR and immunoblottingCC BY · PubMed Central
Fig. 3 β-adrenergic signalling regulates the migration and invasion potential of GC cells in vitro. a–d Stress hormones and specific ADRB2 agonists promoted the migration of GC cells in a wound-healing assay. Propranolol and a specific ADRB2 antagonist attenuated wound healing in HGC27 and MGC803 cells. Original magnification, ×40; Scale bar = 100 μm. Photographs were taken immediately (0 h) and 48 h after wounding, and quantification of wound closure was performed. e, f An invasion assay was performed on each cell group. Original magnification, ×100; Scale bar = 100 μm
Fig. 3 β-adrenergic signalling regulates the migration and invasion potential of GC cells in vitro. a–d Stress hormones and specific ADRB2 agonists promoted the migration of GC cells in a wound-healing assay. Propranolol and a specific ADRB2 antagonist attenuated wound healing in HGC27 and MGC803 cells. Original magnification, ×40; Scale bar = 100 μm. Photographs were taken immediately (0 h) and 48 h after wounding, and quantification of wound closure was performed. e, f An invasion assay was performed on each cell group. Original magnification, ×100; Scale bar = 100 μmCC BY · PubMed Central
Fig. 4 The effects of different subtypes of ADRB agonists and antagonists on cell cycle distribution and apoptosis of HGC27 GC cells. a The ADRB2-blocking group showed a specific loss of the ADRB2 signal in HGC27 cells, and the percentage of apoptotic cells was obviously increased, compared with those of the control and ADRB1-blocking groups. Furthermore, selective activation of ADRB2 significantly reduced the rate of apoptosis. b The percentages of G0-G1 phase cells in the population of cells treated with the specific ADRB2 antagonist ICI118,551 and non-specific ADRB antagonist propranolol were increased compared with those in cells treated with atenolol and the control. Moreover, the percentages of S phase cells were significantly reduced (p < 0.05)
Fig. 4 The effects of different subtypes of ADRB agonists and antagonists on cell cycle distribution and apoptosis of HGC27 GC cells. a The ADRB2-blocking group showed a specific loss of the ADRB2 signal in HGC27 cells, and the percentage of apoptotic cells was obviously increased, compared with those of the control and ADRB1-blocking groups. Furthermore, selective activation of ADRB2 significantly reduced the rate of apoptosis. b The percentages of G0-G1 phase cells in the population of cells treated with the specific ADRB2 antagonist ICI118,551 and non-specific ADRB antagonist propranolol were increased compared with those in cells treated with atenolol and the control. Moreover, the percentages of S phase cells were significantly reduced (p < 0.05)CC BY · PubMed Central
Fig. 5 Chronic restraint stress protocol. a Seven days before the MGC803 cells were injected into the flanks of nude mice, the mice were exposed to chronic stress conditions (daily restraint) vs home cage control conditions for 8 h daily, lasting for 28 days. b Representative tumour images from the chronic stress group and the stress-free control group are displayed. c, d After 4 weeks, the adrenal gland diameters of the control group and the stress group were measured. e The weight curve of the two groups of nude mice is shown. f The mean tumour weight was monitored. g The volume of the tumour during the process of tumour growth at six different time points was determined. h Adrenal gland diameter quantitative results are shown. i, j The average levels of NA and Adr were analysed by ELISA
Fig. 5 Chronic restraint stress protocol. a Seven days before the MGC803 cells were injected into the flanks of nude mice, the mice were exposed to chronic stress conditions (daily restraint) vs home cage control conditions for 8 h daily, lasting for 28 days. b Representative tumour images from the chronic stress group and the stress-free control group are displayed. c, d After 4 weeks, the adrenal gland diameters of the control group and the stress group were measured. e The weight curve of the two groups of nude mice is shown. f The mean tumour weight was monitored. g The volume of the tumour during the process of tumour growth at six different time points was determined. h Adrenal gland diameter quantitative results are shown. i, j The average levels of NA and Adr were analysed by ELISACC BY · PubMed Central
Fig. 6 Chronic stress promotes gastric cancer progression. a MGC803-inoculated mice were treated with PBS, epinephrine, norepinephrine, or propranolol added to drinking water. Another four groups of mice were treated with chronic stress combined with drug treatment. b HGC27 cells were inoculated into the right flanks of nude mice 7 days after initiating stress. c Immunohistochemical staining targeting Ki-67. Original magnification, ×100; Scale bar = 100 μm. d TUNEL assay was used to detect apoptotic cells in tissues. Scale bar = 100 μm. e The body weight of each mouse in each experimental group was recorded during the trial. f Tumour volumes on the indicated days are presented as growth curves. g The mean tumour weight of each group was determined. h The data represent the average serum cortisone levels in each group. i The percentage of cells positive for Ki-67 staining is presented as a Ki-67 index. j Representative TUNEL-positive cells were used to draw the histogram
Fig. 6 Chronic stress promotes gastric cancer progression. a MGC803-inoculated mice were treated with PBS, epinephrine, norepinephrine, or propranolol added to drinking water. Another four groups of mice were treated with chronic stress combined with drug treatment. b HGC27 cells were inoculated into the right flanks of nude mice 7 days after initiating stress. c Immunohistochemical staining targeting Ki-67. Original magnification, ×100; Scale bar = 100 μm. d TUNEL assay was used to detect apoptotic cells in tissues. Scale bar = 100 μm. e The body weight of each mouse in each experimental group was recorded during the trial. f Tumour volumes on the indicated days are presented as growth curves. g The mean tumour weight of each group was determined. h The data represent the average serum cortisone levels in each group. i The percentage of cells positive for Ki-67 staining is presented as a Ki-67 index. j Representative TUNEL-positive cells were used to draw the histogramCC BY · PubMed Central
Fig. 7 In vivo analysis of ADRB2-mediated regulation of GC growth and metastasis. a The relative expression of ADRB2 in gastric cancer tissues compared with that of the corresponding adjacent normal tissues. ADRB2 expression was examined by qPCR and normalized to GAPDH expression. b The expression of ADRB2 was detected in 100 paired GC tissues and adjacent tissues. c Box plot showing the IHC scores for ADRB2 protein expression in human GC tissues. d Immunohistochemical staining of ADRB2 in human normal gastric tissue and tumour tissues. e Photographs of tumours were taken by the IVIS Imaging System. f, g, h, i, j The expression of VEGF, MMP-2, MMP-7 and MMP-9 in tumour samples. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, or ***p < 0.001
Fig. 7 In vivo analysis of ADRB2-mediated regulation of GC growth and metastasis. a The relative expression of ADRB2 in gastric cancer tissues compared with that of the corresponding adjacent normal tissues. ADRB2 expression was examined by qPCR and normalized to GAPDH expression. b The expression of ADRB2 was detected in 100 paired GC tissues and adjacent tissues. c Box plot showing the IHC scores for ADRB2 protein expression in human GC tissues. d Immunohistochemical staining of ADRB2 in human normal gastric tissue and tumour tissues. e Photographs of tumours were taken by the IVIS Imaging System. f, g, h, i, j The expression of VEGF, MMP-2, MMP-7 and MMP-9 in tumour samples. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, or ***p < 0.001CC BY · PubMed Central
Fig. 8 Schematic representation of the proposed mechanism of ADRB2-modulated GC progression and metastasis
Fig. 8 Schematic representation of the proposed mechanism of ADRB2-modulated GC progression and metastasisCC BY · PubMed Central

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  1. Guo, Y. et al. Role of the autonomic nervous system in the tumor micro-environment and its therapeutic potential. Curr. Pharm. Des. 23, 1687–1692 (2017). · DOI

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Metadata

Authors
Zhang, X., Zhang, Y., He, Z., Yin, K., Li, B., Zhang, L., & Xu, Z.
Affiliations
  • Affiliated Hospital of Xuzhou Medical College (CN)
  • Nanjing University of Chinese Medicine (CN)
  • Jiangsu Province Hospital (CN)
  • Affiliated Hospital of Jiangsu University (CN)
Corresponding author
Xu, Zekuan
ORCID
Venue
Cell Death & Disease
Publisher
Springer Science and Business Media LLC
Published
2019-10-17
Volume
10
Issue
11
Pages
788-788
ISSN
2041-4889 (ISSN-L)
DOI
10.1038/s41419-019-2030-2
PMC
PMC6797812
OpenAlex
W2981115917
Cited by
261
References
46
Citations by year
2026: 43 · 2025: 48 · 2024: 32 · 2023: 34 · 2022: 55 · 2021: 32 · 2020: 16
FWCI
20.51
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Death and Disease (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Natural Science Foundation of China (JX10231801, 81272712, 81572362, 20181119, BRA2015474, 81602080, 81272712, 81572362)
  • Government of Jiangsu Province (333 Project)
  • Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, JX10231801, JX 10231801, 81572362, BRA2015474)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Stress, Anesthesia, and Immune Response
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
  • Health Sciences › Medicine › Oncology › Gastric Cancer Management and Outcomes
Keywords
chronic stress, gastric cancer, ADRB2, beta2-adrenergic receptor, adrenergic signaling, tumor progression, metastasis, chronic restraint stress, catecholamines, cortisol, propranolol, atenolol, NF-κB, STAT3, vascular endothelial growth factor, matrix metalloproteinases, cell proliferation, cell invasion
MeSH terms
Animals, Cell Movement, Female, Humans, Male, Mice, Inbred BALB C, Mice, Nude, Neoplasm Metastasis, RNA, Messenger, Stomach Neoplasms, Stress, Physiological, Signal Transduction, NF-kappa B, Receptors, Adrenergic, beta-2, Disease Progression, MAP Kinase Signaling System, Cell Proliferation, STAT3 Transcription Factor, Mice, Heterografts

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