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1.
Onco Targets Ther ; 13: 4665-4678, 2020.
Article in English | MEDLINE | ID: mdl-32547096

ABSTRACT

PURPOSE: To explore the effects of FAM83D on the proliferation, invasion and radiosensitivity of human esophageal cancer cells and to elucidate the mechanism involved in the regulation of the growth and metastasis of esophageal cancer cells. METHODS AND MATERIALS: This study included sixty-nine patients with esophageal cancer. The expression levels of FAM83D in the esophageal cancer tissues and paracarcinoma tissues of the sixty-nine patients were measured. We also examined FAM83D expression in five cell lines. We analyzed the effects of FAM83D on the proliferation, invasion and radiosensitivity of human esophageal cancer cells via MTS, Transwell, and colony formation assays. The effect of FAM83D knockdown on cell apoptosis was assayed by flow cytometry. In addition, we also examined changes in the expression of metastasis-related molecules at the protein and mRNA levels by qRT-PCR and Western blotting after silencing FAM83D expression, and we detected the expression of PI3K/Akt signaling-related proteins by Western blotting. RESULTS: The results demonstrated that the expression of FAM83D was obviously higher in esophageal cancer tissues and cell lines than that in human adjacent normal tissues and normal esophageal epithelial cell lines. FAM83D overexpression was positively associated with tumor size, tumor-node-metastasis (TNM) stage, T classification, N classification, distant metastasis and relapse and was negatively associated with patient survival rates. FAM83D shRNA transfection suppressed its expression. Compared to that in the control group, the proliferation of tumor cells in the FAM83D shRNA group was hindered after exposure to radiation in vitro and in vivo; in addition, FAM83D knockdown inhibited cell invasion, induced apoptosis and regulated apoptosis-related protein expression. Moreover, the radiosensitivity of esophageal cancer cells was increased after depletion of FAM83D. In addition, FAM83D silencing was associated with the reversion of EMT, as reflected by an increase in the epithelial marker E-cadherin and a decrease in the mesenchymal markers N-cadherin and vimentin. Further study showed that FAM83D depletion suppressed the signaling pathway involving p-Akt, p-GSK-3ß and Snail. CONCLUSION: The results reveal that FAM83D may be a potential therapeutic target for esophageal squamous cell carcinoma (ESCC) and that lower expression of FAM83D in coordination with irradiation promotes the radiosensitization of ESCC by inducing EMT through the Akt/GSK-3ß/Snail signaling pathway.

2.
Oncol Rep ; 39(2): 667-678, 2018 Feb.
Article in English | MEDLINE | ID: mdl-29207170

ABSTRACT

B-cell­specific Moloney murine leukaemia virus integration site-1 (BMI-1) contributes to the growth of tumour cells post-irradiation (IR). The aim of the present study was to characterize the effects of BMI-1 on cell viability, radiosensitivity and its mechanisms of action in oesophageal squamous cell cancer (ESCC). Western blotting and immunohistochemistry were employed to evaluate the protein expression of BMI-1 in ESCC cells and specimens, respectively. Additionally, the protein expression levels of BMI-1, H2AK119ub and γH2AX in ESCC cells were detected following different doses of IR and at different times after IR. The protein expression levels of MDC1 and 53BP1 were also measured. Flow cytometry and MTT assays were used to determine cell cycle progression, apoptosis and cell viability. The phosphatidylinositol 3-kinase inhibitor LY294002 and the agonist IGF-1 were employed to suppress or induce the phosphorylation of Akt to determine whether BMI-1 induces radioresistance in ESCC cells via activation of the PI3K/Akt pathway. The expression of BMI-1 was higher in ESCC tissues and cells compared with that in normal oesophageal tissues and cells. In addition, BMI-1 was positively related to tumour size and lymph node metastases and negatively to the overall survival of ESCC patients. IR induced the expression of BMI-1, H2AK119ub and γH2AX in a dose- and time-dependent manner. BMI-1 knockdown lowered the expression of γH2AX, MDC1 and 53BP1, suppressed cell viability and increased radiosensitivity. G2/M phase arrest was eliminated; this was followed by an increased proportion of cells entering the G0/G1 phase after IR and BMI-1 knockdown via the upregulation of P16 and downregulation of cyclin D2 and cyclin-dependent kinase-4. Moreover, BMI-1 knockdown increased cell apoptosis, downregulated MCL-1 and p-Akt and upregulated Bax. Additionally, the inhibitory effect of the downregulation of p-Akt by LY294002 on tumour cell viability was identical to that of BMI-1 knockdown, while the kinase agonist IGF-1 reversed the effects of BMI-1 knockdown on cell viability and radiosensitivity. Taken together, BMI-1 knockdown induces radiosensitivity in ESCC and significantly inhibits cell viability, which may contribute to an increased proportion of cells in the G0/G1 phase and cell apoptosis via suppression of the PI3K/Akt signalling pathway.


Subject(s)
Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/radiotherapy , Esophageal Neoplasms/pathology , Esophageal Neoplasms/radiotherapy , Gene Knockdown Techniques , Polycomb Repressive Complex 1/metabolism , Radiation Tolerance , Adaptor Proteins, Signal Transducing , Animals , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Cell Cycle/radiation effects , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation/radiation effects , Cell Survival/radiation effects , Esophageal Neoplasms/genetics , Esophageal Neoplasms/metabolism , Esophageal Squamous Cell Carcinoma , Female , Gene Expression Regulation, Neoplastic/radiation effects , Histones/metabolism , Humans , Male , Mice , Neoplasm Transplantation , Nuclear Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Polycomb Repressive Complex 1/genetics , Prognosis , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/radiation effects , Trans-Activators/metabolism , Tumor Suppressor p53-Binding Protein 1/metabolism , Up-Regulation/radiation effects
3.
Sci Rep ; 6: 31315, 2016 08 09.
Article in English | MEDLINE | ID: mdl-27501997

ABSTRACT

p-Hydroxylcinnamaldehyde (CMSP) has been identified as an inhibitor of the growth of various cancer cells. However, its function in oesophageal squamous cell carcinoma (ESCC) and the underlying mechanism remain unclear. The aim of the present study was to characterize the differentiation effects of CMSP, as well as its mechanism in the differentiation of ESCC Kyse30 and TE-13 cells. The function of CMSP in the viability, colony formation, migration and invasion of Kyse30 and TE-13 cells was determined by MTS, colony-formation, wound healing and transwell assays. Western blotting and pull-down assays were used to investigate the effect of CMSP on the expression level of malignant markers of ESCC, as well as the activity of MAPKs, RhoA and GTP-RhoA in Kyse30 and TE-13 cells. We found that CMSP could inhibit proliferation and migration and induce Kyse30 and TE-13 cell differentiation, characterized by dendrite-like outgrowth, decreased expression of tumour-associated antigens, as well as the decreased expression of malignant markers. Furthermore, increased cAMP, p-P38 and decreased activities of ERK, JNK and GTP-RhoA, were detected after treatment with CMSP. These results indicated that CMSP induced the differentiation of Kyse30 and TE-13 cells through mediating the cAMP-RhoA-MAPK axis, which might provide new potential strategies for ESCC treatment.


Subject(s)
Acrolein/analogs & derivatives , Carcinoma, Squamous Cell/metabolism , Cinnamates/pharmacology , Esophageal Neoplasms/metabolism , Acrolein/pharmacology , Animals , Biomarkers, Tumor/metabolism , Cell Differentiation , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cell Survival , Cyclic AMP/metabolism , Esophageal Squamous Cell Carcinoma , Esophagus/metabolism , Humans , MAP Kinase Signaling System , Medicine, Chinese Traditional , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , rhoA GTP-Binding Protein/metabolism
4.
Oncol Rep ; 35(6): 3669-78, 2016 Jun.
Article in English | MEDLINE | ID: mdl-27108688

ABSTRACT

Radiotherapy (RT) has been widely used to treat cancer patients, particularly esophageal cancer patients. B-cell-specific Moloney murine leukemia virus integration site-1 (BMI-1) plays an important role in promoting the growth of cancer cells after exposure to irradiation. The present study aimed to characterize the effects of BMI-1 on the proliferation and invasion of cancer cells, as well as the mechanism involved in the regulation of the growth of esophageal cancer ECA109 and TE13 cells. The expression levels of the BMI-1 gene and protein in esophageal cancer ECA109 and TE13 cells were determined by quantitative PCR and western blotting after transfection. Co-immunoprecipitation (Co-IP) assay was employed to detect the interaction of BMI-1 with r-H2AX and H2AK119ub. We used flow cytometry to analyze the cell cycle distribution and apoptosis of transfected cells after irradiation or not, and examined cellular growth and invasion in vitro by MTS and Transwell assays. The results revealed that shRNA targeting the BMI-1 gene and protein downregulated BMI-1 expression after transfection for 24 h. The proliferation and invasion of tumor cells in the BMI-1­shRNA group were suppressed after RT. In addition, the interaction of BMI-1, H2AK119ub and r-H2AX was increased after exposure to IR, followed by an increased apoptosis rate and decreased percentage of cells arrested at the G2/M phase after irradiation and silencing of BMI-1 by shRNA. Knockdown of BMI-1 expression decreased the phosphorylation of H2AX, upregulated p16, and induced the radiosensitivity of esophageal cancer ECA109 and TE13 cells in vitro and significantly inhibited the growth and invasion of tumor cells. The mechanisms were found to be abrogation of cell cycle arrest at the G2/M stage and promotion of apoptosis.


Subject(s)
Apoptosis/radiation effects , Carcinoma, Squamous Cell/radiotherapy , Esophageal Neoplasms/radiotherapy , G2 Phase Cell Cycle Checkpoints/genetics , Polycomb Repressive Complex 1/genetics , Radiation Tolerance/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cell Proliferation/genetics , Cyclin-Dependent Kinase Inhibitor p16/biosynthesis , Esophageal Neoplasms/genetics , Esophageal Neoplasms/pathology , Flow Cytometry , Gene Expression Regulation, Neoplastic , Histones/metabolism , Humans , Neoplasm Invasiveness/genetics , Phosphorylation/genetics , Polycomb Repressive Complex 1/metabolism , Proto-Oncogene Proteins c-bcl-2/biosynthesis , RNA Interference , RNA, Small Interfering/genetics , Radiation Tolerance/drug effects
5.
Int J Oncol ; 48(5): 1985-96, 2016 May.
Article in English | MEDLINE | ID: mdl-26936624

ABSTRACT

Radiotherapy has been widely used for the treatment of cancer patients, especially for esophageal cancer patients. Ring finger protein 2 (RNF2) plays an important role in promoting the growth of cancer cells after exposure to irradiation. The present study aims to characterize the proliferative effects of RNF2 on cancer cells, and its mechanisms on the growth of esophageal cancer cells. We demonstrate that expression of RNF2 was markedly upregulated in esophageal cancer cell lines and surgically resected cancer specimens. In addition, RNF2 expression level is positively correlated with the presence of tumor size, lymph node metastases and negatively correlated with patient survival rates, suggesting that it plays an important role in the progression of esophageal cancer. Furthermore, the expression of RNF2 at both mRNA and protein levels in esophageal cancer ECA109 and TE13 cells was detected by real-time PCR and western blot assay after shRNA targeting RNF2. Co-immunoprecipitation (Co-IP) assay and western blot analysis were employed to detect the interaction between RNF2 and r-H2AX, H2AK119ub, and the expression of proteins associated with cell cycle and apoptosis, respectively. We used flow cytometry assay to analyze cell cycle and apoptosis of transfected cells, and further examined cellular growth in vitro and in vivo. shRNA targeting RNF2 gene and protein downregulated RNF2 expression after transfection for 24 h. The proliferation of tumor cells in RNF2-shRNA group was suppressed after radiotherapy. In addition, the interaction of RNF2, H2AK119ub, r-H2AX was increased after exposure to IR, followed by increasing apoptosis rates and inducing the arrest at G0/G1 phase after irradiation and shRNA targeting RNF2. Expression of the short-hairpin RNA is also correlated with the upregulation of p16 and Bax, and the downregulation of cyclin D2, cyclin-dependent kinase (CDK)-4, H2AX and Bcl-2. RNF2 gene knockdown induces radiosensitivity of esophageal cancer cells in vitro and significantly inhibits the growth of tumor cells. The mechanisms include inducing the cell cycle arrest at G0/G1 phase and promoting apoptosis.


Subject(s)
Esophageal Neoplasms/metabolism , Esophageal Neoplasms/pathology , Gene Knockdown Techniques/methods , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 1/metabolism , Apoptosis , Cell Cycle Checkpoints/radiation effects , Cell Line, Tumor , Cell Proliferation/radiation effects , Cell Survival/radiation effects , Esophageal Neoplasms/genetics , Esophageal Neoplasms/radiotherapy , Female , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Male , Prognosis , Survival Analysis , Up-Regulation
6.
Zhongguo Zhong Yao Za Zhi ; 40(6): 1207-11, 2015 Mar.
Article in Chinese | MEDLINE | ID: mdl-26226772

ABSTRACT

To investigate the effect of Tanreqing injection on immune activity of peripheral blood lymphocytes of patients with lung cancer. The peripheral blood lymphocytes of patients with lung cancer and healthy persons were separated by the density gradient centrifugation method for subsequent experiments, with those from healthy persons as the positive control. The effect of Tanreqing injection on stimulating the proliferation of lymphocytes with phytohemagglutinin (PHA) was determined by MTT method. The effect of Tanreqing injection on the lymphocyte secretions of IFN-γ and TNF-α and the subset ratio of lymphocytes cultured separately or with Tanreqing injection of different concentrations were examined by ELISA and flow cytometry (FCM) respectively. In addition, the LDH release assay was used to detect the cytotoxicity of cytotoxic T cells (CTL) and natural killer cells (NK). According to the findings, all of immunological indexes of lymphocytes from patients with lung cancer were weaker than that of healthy persons, but with the obvious increases in proliferation activity and IFN-γ and TNF-α secretions of lymphocytes co-cultured with Tanreqing Injection (P < 0.05). Among lymphocyte subsets co-cultured with Tanreqing Injection, CD3+, CD3+ CD4+ and CD3- CD16 + 56+ cell ratios notably increased, whereas CD4+ CD25+ Treg cell ratio obviously decreased (P < 0.05). In the meantime, Tanreqing injection can markedly promote the cytotoxicities of CTL and NK (P < 0.05). In conclusion, Tanreqing injection shows a significant effect in promoting the immune activity of lymphocytes from patients with lung cancer and their anti-tumor immunity.


Subject(s)
Drugs, Chinese Herbal/administration & dosage , Killer Cells, Natural/immunology , Lung Neoplasms/drug therapy , Lung Neoplasms/immunology , T-Lymphocytes, Cytotoxic/immunology , Cell Proliferation/drug effects , Cells, Cultured , Humans , Interferon-gamma/genetics , Interferon-gamma/immunology , Killer Cells, Natural/drug effects , Lung Neoplasms/genetics , Lung Neoplasms/physiopathology , T-Lymphocytes, Cytotoxic/drug effects , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology
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