Mol. Cells 2021; 44(4): 267-278
Published online April 6, 2021
https://doi.org/10.14348/molcells.2021.2179
© The Korean Society for Molecular and Cellular Biology
Correspondence to : yelin@hospital.cqmu.edu.cn
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Metallothionein (MT1M) belongs to a family of cysteine-rich cytosolic protein and has been reported to be a tumor suppressor gene in multiple cancers. However, its role in esophageal carcinoma carcinogenesis remains unclear. In this study, MT1M expression was correlated with tumor type, stage, drinking and smoking history, as well as patient survival. We also studied the regulation and biological function of MT1M in esophageal squamous cell carcinoma (ESCC). We have found that MT1M is significantly downregulated in ESCC tissues compared with adjacent non-cancer tissues. Furthermore, restoration of expression by treatment with the demethylation agent A + T showed that MT1M downregulation might be closely related to hypermethylation in its promoter region. Over-expression of MT1M in ESCC cells significantly altered cell morphology, induced apoptosis, and reduced colony formation, cell viability, migration and epithelial-mesenchymal transition. Moreover, based on reactive oxygen species (ROS) levels, a superoxide dismutase 1 (SOD1) activity assay and protein analysis, we verified that the tumor-suppressive function of MT1M was at least partially caused by its upregulation of ROS levels, downregulation of SOD1 activity and phosphorylation of the SOD1 downstream pathway PI3K/AKT. In conclusion, our results demonstrated that MT1M was a novel tumor-suppressor in ESCC and may be disrupted by promoter CpG methylation during esophageal carcinogenesis.
Keywords esophageal squamous cell carcinoma, MT1M, PI3K/AKT, SOD1, tumor suppressor
China has the highest incidence of esophageal cancer worldwide. This type of cancer consists of two main histological types: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) (Siegel et al., 2020). In Asian countries, especially China, 90% of esophageal cancer cases are identified as ESCC. In low-risk areas, EAC is the most common histologic type (Zeng et al., 2016). Therefore, understanding the mechanisms of ESCC development is of great interest in China. The molecular mechanisms of esophageal carcinogenesis are not clear, but multiple step processes and very complex signaling pathways are involved. Silencing of tumor suppressor genes (TSGs) by genetic and epigenetic pathways has recently been revealed as an important step in esophageal tumorigenesis (Cheng et al., 2012; Ye et al., 2019). Understanding the function and mechanisms of these TSGs in esophageal cancer will help us to find molecular biomarkers to predict the occurrence and outcome of esophageal cancer earlier. Biomarkers for EAC are currently a hotspot for research. However, ESCC is not as well studied as EAC in terms of TSGs. The molecular biomarkers used clinically in ESCC diagnosis are rare. So, study of the function and regulation of silencing TSGs in ESCC is urgently needed.
Metallothioneins (
Recently, our group found that in the UALCAN database
Esophageal cancer cell lines KYSE150, KYSE510, KYSE960 (cell lines were gifts from Chongqing Key Laboratory of Molecular Oncology and Epigenetics, The First Affiliated Hospital of Chongqing Medical University, cells were originally from the Japanese Cancer Research Resources Bank, Japan) were used in this study. Cell lines were maintained in a 1:1 combination of Ham’s F12 and RPMI 1640 media (Gibco-BRL, Germany) with 10% fetal bovine serum (FBS; PAA Laboratories, Austria) and 1% penicillin, streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. 293T human epithelial cells and BEAS-2B cells were cultured in DMEM with 10% FBS. 293T cell and BEAS-2B cells are used as a positive control.
Fresh NSCLC tissues and tumor-adjacent tissues were obtained from patients who underwent esophagectomy at the Department of Cardiothoracic Surgery in the First Affiliated Hospital of Chongqing Medical University (Patient clinical features were listed in Table 1). This research was approved by the Institutional Ethics Committees of the First Affiliated Hospital of Chongqing Medical University (No. 2019-11) and followed the principles of the Declaration of Helsinki. Patient consent forms were signed by each patient who participated in this study.
The UALCAN database (http://ualcan.path.uab.edu/) was used to analyze the correlation between
Cell lines and tissue samples treated with DNase I was used for RNA was extraction by TRIzol reagent (Molecular Research Center, USA). The RNA concentrations were measured by spectrophotometry and the store temperature was –80°C. By Promega Reverse Transcription System (Promega, USA) RNA was reversely transcribed. Semi-quantitative polymerase chain reaction (PCR) was carried out using Go-Taq DNA polymerase (Promega) and reaction conditions were as we reported before (Ye et al., 2018), GAPDH were used as internal control. Real-time PCR used ABI SYBR green on an ABI 7500 real-time PCR detection system (Applied Biosystems, USA) and conditions were as reported (Ye et al., 2018). GAPDH was used as a loading control.
The primers used were as follows;
KYSE 150 and KYSE960 cell lines (1 × 105 cells/ml) were seeded in 100 mm dishes. As soon as cells settling down, DNA demethylating agent 5-Aza (Sigma-Aldrich, USA) and histone deacetylase inhibitor trichostatin A (TSA; Cayman Chemical, USA) were applied as previously described (Ye et al., 2019). Cells were collected 24 h after TSA treatment and
pcDNA3.1(+)-Flag-
KYSE150 and KYSE960 cells (800 cells/well) stably transfected with pcDNA3.1-
Cell proliferation was carried out by MTS assay. Stable
Cell migration ability was also demonstrated using Transwell chambers (8-μm pore size; Corning, USA). For migration assay, KYSE150 and KYSE960 cells stably transfected with pcDNA3.1-
For apoptosis, KYSE150 and KYSE960 cells were transiently transfected with 4 μg pcDNA3.1-
Stable
Cell lines stable expressing vector or pcDNA3.1-
Cells were cultured on coverslips and transfected with
Cell lines stable expressing vector or pcDNA3.1-
To detect the effect of ZnSO4 supplementation on
Intracellular ROS levels were measured using 2’,7’-dichlorofluorescin diacetate (H2DCFDA; Invitrogen) as described in the instructions. Generally, cells stably expressing vector and
All data are representative of three independent experiments and presented as mean ± SD. SPSS software (ver. 16.0; SPSS, USA) was used for statistical analyses. Student’s
To understand the function of
To further analyze whether its downregulation was correlated with
To assess whether there was any correlation between
Together, these findings indicated that the downregulation of
In order to verify whether
To investigate the impact of
In order to further explore the tumor suppressive effects of
Taken together, through the study of cellular function, we have shown that
Moreover, to further demonstrate whether
In humans,
The present report aimed to study the role of
To understand the exact mechanisms of
Phosphatidylinositol 3-kinase-Akt (PI3K-Akt), mitogen-activated protein kinase (MAPK), AMP-activated protein kinase (AMPK), nuclear factor-κB (NF-κB), and c-Jun N-terminal kinase (JNK) are the downstream signaling pathways regulated by the
In conclusion, in this study we identified
This study was supported by Natural Science Foundation of Chongqing, Commission of Science and Technology of Chongqing, China (cstc2019jcyj-msxmX0861).
D.L., W.P., and B.W. performed experiments and analyzed data. H.L. and R.Z. (Ruizhen Zhang) prepared and provided samples and reagents. R.Z. (Ruiqin Zhou) and L.Y. (Lijun Yao) collected patient samples and recorded patient information. L.Y. (Lin Ye) designed and supported the whole study and wrote the manuscript.
The authors have no potential conflicts of interest to disclose.
Clinicopathological features of 15 ESCC patients
Clinicopathological features | No. of patients (n = 15) |
---|---|
Sex | |
Male | 11 |
Female | 4 |
Age | |
≤ 50 y | 3 |
51-59 y | 8 |
≥ 60 y | 4 |
Phase | |
I | 5 |
II | 7 |
III | 3 |
IV | 0 |
Tumor size | |
< 3.0 cm | 4 |
3.0 to < 5.0 cm | 10 |
5.0 to < 7.0 cm | 1 |
≥ 7.0 cm | 0 |
Lymph node metastasis | |
Present | 12 |
Absent | 3 |
Distant metastasis | |
Present | 2 |
Absent | 13 |
Smoking history | |
Smoker | 11 |
Non-smoker | 4 |
Drinking history | |
Drinking | 9 |
Never-drinking | 6 |
Tumor histology | |
Adenocarcinoma | 0 |
Squamous cell carcinoma | 15 |
Mol. Cells 2021; 44(4): 267-278
Published online April 30, 2021 https://doi.org/10.14348/molcells.2021.2179
Copyright © The Korean Society for Molecular and Cellular Biology.
Dandan Li1,2 , Weiyan Peng3
, Bin Wu5
, Huan Liu4
, Ruizhen Zhang2
, Ruiqin Zhou4
, Lijun Yao4
, and Lin Ye4,*
1Laboratory Research Center, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China, 2Department of Otolaryngology Head and Neck Surgery, Daping Hospital, Army Medical University, Chongqing 400042, China, 3Chongqing Key Laboratory of Molecular Oncology and Epigenetics, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China, 4Department of Cardiothoracic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China, 5Department of Anesthesiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
Correspondence to:yelin@hospital.cqmu.edu.cn
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Metallothionein (MT1M) belongs to a family of cysteine-rich cytosolic protein and has been reported to be a tumor suppressor gene in multiple cancers. However, its role in esophageal carcinoma carcinogenesis remains unclear. In this study, MT1M expression was correlated with tumor type, stage, drinking and smoking history, as well as patient survival. We also studied the regulation and biological function of MT1M in esophageal squamous cell carcinoma (ESCC). We have found that MT1M is significantly downregulated in ESCC tissues compared with adjacent non-cancer tissues. Furthermore, restoration of expression by treatment with the demethylation agent A + T showed that MT1M downregulation might be closely related to hypermethylation in its promoter region. Over-expression of MT1M in ESCC cells significantly altered cell morphology, induced apoptosis, and reduced colony formation, cell viability, migration and epithelial-mesenchymal transition. Moreover, based on reactive oxygen species (ROS) levels, a superoxide dismutase 1 (SOD1) activity assay and protein analysis, we verified that the tumor-suppressive function of MT1M was at least partially caused by its upregulation of ROS levels, downregulation of SOD1 activity and phosphorylation of the SOD1 downstream pathway PI3K/AKT. In conclusion, our results demonstrated that MT1M was a novel tumor-suppressor in ESCC and may be disrupted by promoter CpG methylation during esophageal carcinogenesis.
Keywords: esophageal squamous cell carcinoma, MT1M, PI3K/AKT, SOD1, tumor suppressor
China has the highest incidence of esophageal cancer worldwide. This type of cancer consists of two main histological types: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) (Siegel et al., 2020). In Asian countries, especially China, 90% of esophageal cancer cases are identified as ESCC. In low-risk areas, EAC is the most common histologic type (Zeng et al., 2016). Therefore, understanding the mechanisms of ESCC development is of great interest in China. The molecular mechanisms of esophageal carcinogenesis are not clear, but multiple step processes and very complex signaling pathways are involved. Silencing of tumor suppressor genes (TSGs) by genetic and epigenetic pathways has recently been revealed as an important step in esophageal tumorigenesis (Cheng et al., 2012; Ye et al., 2019). Understanding the function and mechanisms of these TSGs in esophageal cancer will help us to find molecular biomarkers to predict the occurrence and outcome of esophageal cancer earlier. Biomarkers for EAC are currently a hotspot for research. However, ESCC is not as well studied as EAC in terms of TSGs. The molecular biomarkers used clinically in ESCC diagnosis are rare. So, study of the function and regulation of silencing TSGs in ESCC is urgently needed.
Metallothioneins (
Recently, our group found that in the UALCAN database
Esophageal cancer cell lines KYSE150, KYSE510, KYSE960 (cell lines were gifts from Chongqing Key Laboratory of Molecular Oncology and Epigenetics, The First Affiliated Hospital of Chongqing Medical University, cells were originally from the Japanese Cancer Research Resources Bank, Japan) were used in this study. Cell lines were maintained in a 1:1 combination of Ham’s F12 and RPMI 1640 media (Gibco-BRL, Germany) with 10% fetal bovine serum (FBS; PAA Laboratories, Austria) and 1% penicillin, streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. 293T human epithelial cells and BEAS-2B cells were cultured in DMEM with 10% FBS. 293T cell and BEAS-2B cells are used as a positive control.
Fresh NSCLC tissues and tumor-adjacent tissues were obtained from patients who underwent esophagectomy at the Department of Cardiothoracic Surgery in the First Affiliated Hospital of Chongqing Medical University (Patient clinical features were listed in Table 1). This research was approved by the Institutional Ethics Committees of the First Affiliated Hospital of Chongqing Medical University (No. 2019-11) and followed the principles of the Declaration of Helsinki. Patient consent forms were signed by each patient who participated in this study.
The UALCAN database (http://ualcan.path.uab.edu/) was used to analyze the correlation between
Cell lines and tissue samples treated with DNase I was used for RNA was extraction by TRIzol reagent (Molecular Research Center, USA). The RNA concentrations were measured by spectrophotometry and the store temperature was –80°C. By Promega Reverse Transcription System (Promega, USA) RNA was reversely transcribed. Semi-quantitative polymerase chain reaction (PCR) was carried out using Go-Taq DNA polymerase (Promega) and reaction conditions were as we reported before (Ye et al., 2018), GAPDH were used as internal control. Real-time PCR used ABI SYBR green on an ABI 7500 real-time PCR detection system (Applied Biosystems, USA) and conditions were as reported (Ye et al., 2018). GAPDH was used as a loading control.
The primers used were as follows;
KYSE 150 and KYSE960 cell lines (1 × 105 cells/ml) were seeded in 100 mm dishes. As soon as cells settling down, DNA demethylating agent 5-Aza (Sigma-Aldrich, USA) and histone deacetylase inhibitor trichostatin A (TSA; Cayman Chemical, USA) were applied as previously described (Ye et al., 2019). Cells were collected 24 h after TSA treatment and
pcDNA3.1(+)-Flag-
KYSE150 and KYSE960 cells (800 cells/well) stably transfected with pcDNA3.1-
Cell proliferation was carried out by MTS assay. Stable
Cell migration ability was also demonstrated using Transwell chambers (8-μm pore size; Corning, USA). For migration assay, KYSE150 and KYSE960 cells stably transfected with pcDNA3.1-
For apoptosis, KYSE150 and KYSE960 cells were transiently transfected with 4 μg pcDNA3.1-
Stable
Cell lines stable expressing vector or pcDNA3.1-
Cells were cultured on coverslips and transfected with
Cell lines stable expressing vector or pcDNA3.1-
To detect the effect of ZnSO4 supplementation on
Intracellular ROS levels were measured using 2’,7’-dichlorofluorescin diacetate (H2DCFDA; Invitrogen) as described in the instructions. Generally, cells stably expressing vector and
All data are representative of three independent experiments and presented as mean ± SD. SPSS software (ver. 16.0; SPSS, USA) was used for statistical analyses. Student’s
To understand the function of
To further analyze whether its downregulation was correlated with
To assess whether there was any correlation between
Together, these findings indicated that the downregulation of
In order to verify whether
To investigate the impact of
In order to further explore the tumor suppressive effects of
Taken together, through the study of cellular function, we have shown that
Moreover, to further demonstrate whether
In humans,
The present report aimed to study the role of
To understand the exact mechanisms of
Phosphatidylinositol 3-kinase-Akt (PI3K-Akt), mitogen-activated protein kinase (MAPK), AMP-activated protein kinase (AMPK), nuclear factor-κB (NF-κB), and c-Jun N-terminal kinase (JNK) are the downstream signaling pathways regulated by the
In conclusion, in this study we identified
This study was supported by Natural Science Foundation of Chongqing, Commission of Science and Technology of Chongqing, China (cstc2019jcyj-msxmX0861).
D.L., W.P., and B.W. performed experiments and analyzed data. H.L. and R.Z. (Ruizhen Zhang) prepared and provided samples and reagents. R.Z. (Ruiqin Zhou) and L.Y. (Lijun Yao) collected patient samples and recorded patient information. L.Y. (Lin Ye) designed and supported the whole study and wrote the manuscript.
The authors have no potential conflicts of interest to disclose.
. Clinicopathological features of 15 ESCC patients.
Clinicopathological features | No. of patients (n = 15) |
---|---|
Sex | |
Male | 11 |
Female | 4 |
Age | |
≤ 50 y | 3 |
51-59 y | 8 |
≥ 60 y | 4 |
Phase | |
I | 5 |
II | 7 |
III | 3 |
IV | 0 |
Tumor size | |
< 3.0 cm | 4 |
3.0 to < 5.0 cm | 10 |
5.0 to < 7.0 cm | 1 |
≥ 7.0 cm | 0 |
Lymph node metastasis | |
Present | 12 |
Absent | 3 |
Distant metastasis | |
Present | 2 |
Absent | 13 |
Smoking history | |
Smoker | 11 |
Non-smoker | 4 |
Drinking history | |
Drinking | 9 |
Never-drinking | 6 |
Tumor histology | |
Adenocarcinoma | 0 |
Squamous cell carcinoma | 15 |
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