J Gastric Cancer.  2020 Mar;20(1):95-105. 10.5230/jgc.2020.20.e9.

The Influence of Bcl-3 Expression on Cell Migration and Chemosensitivity of Gastric Cancer Cells via Regulating Hypoxia-Induced Protective Autophagy

• Hu L
• Bai Z
• Ma X
• Bai N
• Zhang Z

• KMID: 2471929
• DOI: http://doi.org/10.5230/jgc.2020.20.e9

Abstract

PURPOSE
Gastric cancer is a highly metastatic malignant tumor, often characterized by chemoresistance and high mortality. In the present study, we aimed to investigate the role of B-cell lymphoma 3 (Bcl-3) protein on cell migration and chemosensitivity of gastric cancer.
MATERIALS AND METHODS
The gastric cancer cell lines, AGS and NCI-N87, were used for the in vitro studies and the in vivo studies were performed using BALB/c nude mice. Western blotting, wound healing assay, Cell Counting Kit-8 assay, immunohistochemistry, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay were used to evaluate the role of Bcl-3 in gastric cancer.
RESULTS
We found that the protein expression of hypoxia (HYP)-inducible factor-1α and Bcl-3 were markedly upregulated under hypoxic conditions in both AGS and NCI-N87 cells in a time-dependent manner. Interestingly, small interfering RNA-mediated knockdown of Bcl-3 expression affected the migration and chemosensitivity of the gastric cancer cells. AGS and NCI-N87 cells transfected with si-RNA-Bcl-3 (si-Bcl-3) showed significantly reduced migratory ability and increased chemosensitivity to oxaliplatin, 5-fluorouracil, and irinotecan. In addition, si-Bcl-3 restored the autophagy induced by HYP. Further, the protective role of si-Bcl-3 on the gastric cancer cells could be reversed by the autophagy inducer, rapamycin. Importantly, the in vivo xenograft tumor experiments showed similar results.
CONCLUSIONS
Our present study reveals that Bcl-3 knockdown inhibits cell migration and chemoresistance of gastric cancer cells through restoring HYP-induced autophagy.

Keyword

Bcl-3; HIF-1α; Autophagy; Migration; Chemosensitivity

MeSH Terms

Animals
Anoxia
Autophagy*
Blotting, Western
Cell Count
Cell Line
Cell Movement*
DNA Nucleotidylexotransferase
Fluorouracil
Heterografts
Immunohistochemistry
In Vitro Techniques
Lymphoma, B-Cell
Mice
Mice, Nude
Mortality
Sirolimus
Stomach Neoplasms*
Wound Healing
DNA Nucleotidylexotransferase
Fluorouracil
Sirolimus

Figure

• 

  Fig. 1 HYP induces HIF-1α and Bcl-3 protein in gastric cancer cells. (A) Relative expression of HIF-1α in AGS and NCI-N87 cells was analyzed by western blotting. (B) Relative expression of Bcl-3 in AGS and NCI-N87 cells was analyzed by western blotting. GAPDH was used as a loading control.HIF-1α = hypoxia-inducible factor-1α; Bcl-3 = B-cell lymphoma 3; HYP = hypoxia; GAPDH = glyceraldehyde 3-phosphate dehydrogenase.*P<0.05 (compared to control); †P<0.05 (compared to HYP 6 hours); ‡P<0.05 (compared to HYP 12 hours).

• 

  Fig. 2 Knockdown of Bcl-3 reduces the migration and increases the chemosensitivity of gastric cancer cells. (A) AGS and NCI-N87 cells were transfected with si-NC, or si-Bcl-3-1/2/3. Relative expression Bcl-3 in AGS and NCI-N87 cells was analyzed by western blotting. GAPDH was used as a loading control. (B) Cells were treated with HYP/si-Bcl-3 transfection. Wound healing assay was used to assess the migration of AGS and NCI-N87 cells. (C) Quantification of cell migration in AGS and NCI-N87 cells. (D) NCI-N87 cells were treated with or without OXA under HYP and MTT assay was used to detect cell proliferation. (E) NCI-N87 cells were treated with or without 5-FU under HYP and MTT assay was used to detect cell proliferation. (F) NCI-N87 cells were treated with or without IRI under HYP and MTT assay was used to detect cell proliferation.Bcl-3 = B-cell lymphoma 3; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; si-NC = si-negative control; si-Bcl-3 = si-RNA-Bcl-3; HYP = hypoxia; OXA = oxaliplatin; 5-FU = 5-fluorouracil; IRI = irinotecan.*P<0.05 (compared to control); †P<0.05 (compared to HYP); ‡P<0.05 (compared to OXA+HYP); §P<0.05 (compared to 5-FU+HYP); ∥P<0.05 (compared to IRI+HYP).

• 

  Fig. 3 Knockdown of Bcl-3 decreases HYP-induced autophagy. Relative expression of HIF-1α, Atg5, and LC3 were analyzed in AGS and NCI-N87 by western blotting. (B-E) Quantification of the relative protein levels of HIF-1α, Atg5, Bcl-3, and LC3 in AGS and NCI-N87 cells. (F,G) Immunofluorescence assay for LC3. LC3 positive puncta in individual cells are shown.Bcl-3 = B-cell lymphoma 3; HIF-1α = hypoxia-inducible factor-1α; HYP = hypoxia; si-Bcl-3 = si-RNA-Bcl-3; si-NC = si-negative control; GAPDH = glyceraldehyde 3-phosphate dehydrogenase.*P<0.05 (compared to control); †P<0.05 (compared to HYP).

• 

  Fig. 4 Knockdown of Bcl-3 decreases hypoxia-induced autophagy. AGS and NCI-N87 cells were transfected with si-NC/si-Bcl-3 with or without Rapa. (A) Wound healing assay was used to analyze the migration of AGS and NCI-N87 cells. (B) Quantification of cell migration in AGS and NCI-N87 cells. (C) NCI-N87 cells were transfected with si-NC/si-Bcl-3 with or without 5-FU/Rapa, MTT was used to detect cell proliferation. (D) Relative expression of autophagy-related proteins in AGS and NCI-N87 were analyzed by western blotting. (E, F) Quantification of the relative protein levels of Atg5 and LC3 in AGS and NCI-N87 cells.Bcl-3 = B-cell lymphoma 3; si-Bcl-3 = si-RNA-Bcl-3; si-NC = si-negative control; 5-FU = 5-fluorouracil; Rapa = rapamycin; GAPDH = glyceraldehyde 3-phosphate dehydrogenase.*P<0.05 (compared to control); †P<0.05 (compared to si-Bcl-3); ‡P<0.05 (compared to si-Bcl-3+Rapa); §P<0.05 (compared to 5-FU+Rapa); ∥P<0.05 (compared to si-Bcl-3+5-FU);); ¶P<0.05 (compared to si-Bcl-3); **P<0.05 (compared to si-Bcl-3+Rapa).

• 

  Fig. 5 Knockdown of Bcl-3 regulates in vivo tumor growth through autophagy. Tumorigenicity assay of AGS cells were performed using nude mouse. (A) Images of xenograft tumors derived from tumor-bearing nude mice injected with AGS cells with or without si-Bcl-3 transfection and 5-FU/Rapa. Tumor sizes of control and experimental mice are summarized in the line chart. (B-H) Relative expression of the associated proteins. (I) Immunohistochemistry for Ki67 and quantitation of the relative Ki67 expression. (J) TUNEL assay was used to detect apoptosis in the tumor tissue.Bcl-3 = B-cell lymphoma 3; HIF-1α = hypoxia-inducible factor-1α; VEGF = vascular endothelial growth factor; MMP-9 = matrix metallopeptidase 9; NC = negative control; si-Bcl-3 = si-RNA-Bcl-3; 5-FU = 5-fluorouracil; Rapa = rapamycin; TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling; GAPDH = glyceraldehyde 3-phosphate dehydrogenase.*P<0.05 (compared to control); †P<0.05 (compared to si-Bcl-3+5-FU).

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