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. 2018 Apr;109(4):1066-1074.
doi: 10.1111/cas.13523. Epub 2018 Feb 26.

Antitumor effects of metformin are a result of inhibiting nuclear factor kappa B nuclear translocation in esophageal squamous cell carcinoma

Nobufumi Sekino  1 , Masayuki Kano  1 , Yasunori Matsumoto  1 , Haruhito Sakata  1 , Yasunori Akutsu  1 , Naoyuki Hanari  1 , Kentaro Murakami  1 , Takeshi Toyozumi  1 , Masahiko Takahashi  1 , Ryota Otsuka  1 , Masaya Yokoyama  1 , Tadashi Shiraishi  1 , Koichiro Okada  1 , Isamu Hoshino  1 , Keiko Iida  1 , Aki Komatsu Akimoto  1 , Hisahiro Matsubara  1
Affiliations

Antitumor effects of metformin are a result of inhibiting nuclear factor kappa B nuclear translocation in esophageal squamous cell carcinoma

Nobufumi Sekino et al. Cancer Sci. 2018 Apr.

Abstract

Esophageal squamous cell carcinoma (ESCC) is an intractable digestive organ cancer that has proven difficult to treat despite multidisciplinary therapy, and a new treatment strategy is demanded. Metformin is used for type 2 diabetes mellitus and its antitumor effects have been reported recently. Metformin exerts antitumor effects in various respects, such as inhibiting inflammation, tumor growth and epithelial-mesenchymal transition (EMT). However, few reports have described the efficacy of metformin on ESCC, and their findings have been controversial. We analyzed the antitumor effects of metformin and clarified its effects on anti-inflammation, growth suppression and EMT inhibition. Activation of nuclear factor kappa B (NF-κB), the major transcription factor induced by inflammation, was investigated by immunostaining. We found that localization of NF-κB in the nucleus was reduced after metformin treatment. This suggests that metformin inhibited the activation of NF-κB. Metformin inhibited tumor growth and induced apoptosis in ESCC cell lines. Associated with EMT, we examined cell motility by a wound healing assay and the epithelial marker E-cadherin expression of various ESCC cell lines by western blotting. Metformin inhibited cell motility and induced E-cadherin expression. In conclusion, metformin showed multiple antitumor effects such as growth suppression, invasion inhibition, and control of EMT by inhibiting NF-κB localization on ESCC. Further exploration of the marker of treatment efficacy and combination therapy could result in the possibility for novel treatment to use metformin on ESCC.

Keywords: epithelial-mesenchymal transition; esophageal squamous cell carcinoma; inflammation; metformin; treatment outcome.

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Figures

Figure 1
Figure 1
Metformin affects the intracellular localization of nuclear factor kappa B (NF‐κB). A, Western blotting analysis of the NF‐κB protein expression on TE2 cells treated with various concentrations of metformin (0, 0.3, 1, 3 mmol/L) for 24 h. Expression level of β‐actin was used as an endogenous control. B, Local existence of NF‐κB in cells was evaluated by immunostaining. In the metformin group, 0.3 mmol/L metformin was given for 24 h. Images of immunohistochemistry staining with NF‐κB (upper) and the nucleus stained by DAPI (lower) are shown. (upper left, lower left) Control cells. (upper right, lower right) Metformin‐treated cells. Magnification, ×1000. C, Brightness in the cytoplasm and nucleus of each cell was quantified using the Image J program, and the nuclear/cytoplasmic (N/C) ratio was calculated (n = 10). NF‐κB expression in the nucleus was significantly lower in the metformin‐treated group than in the control group (*P = .012)
Figure 2
Figure 2
Proliferation assay and AMP‐activated kinase (AMPK) and liver kinase B1 (LKB1) protein expression in esophageal squamous cell carcinoma (ESCC) cell lines. A, Proliferation assay using Cell Counting Kit‐8 (CCK‐8; Dojindo, Kumamoto, Japan). Metformin was given to ESCC cells for 72 h. B, Western blotting analysis of AMPK and LKB1 protein expression at steady state in these ESCC cell lines
Figure 3
Figure 3
Tumor growth and apoptosis in vivo. Relative tumor volume in the xenograft model of (A) TE2‐FUCCI (n = 7) and (B) TE14 (n = 6). In both the TE2‐FUCCI and TE14 models, relative tumor volume was significantly smaller in the metformin‐treated group than in the control group (TE2‐FUCCI: *P = .033, TE14: **P = .031). Bar, SE. Bodyweight of the xenograft model at days 14 and 42 after the injection of (C) TE2‐FUCCI (n = 7) and (D) TE14 (n = 6) is shown. There were no significant differences between the control group and the metformin‐treated group. Bar, SD. E, In the TE2‐FUCCI xenograft model, the apoptotic cells were detected by TUNEL stain (brown nuclear cell: positive; green nuclear cell: negative). F, Average number of apoptotic cells per view in the TE2‐FUCCI xenograft model (n = 6). There were significantly more apoptotic cells in the tumors in the metformin‐treated group than in the control group (*P = .0257). Bar, SD
Figure 4
Figure 4
Wound healing assay. A, T.Tn and TE2‐FUCCI cells were treated with metformin and captured after 22 h from scratch. Magnification, ×200. Relative distance of the intracellular space is shown (n = 7). Bar, SD. Cell strains: (B) T.Tn, (C) TE2‐FUCCI
Figure 5
Figure 5
Cell migration/invasion assay. A cell migration assay was carried out. The migrating cells were fixed and stained at 24 h after seeding. A, TE2 cells, *P = .01 (0 vs 3 mmol/L), and **P = .01 (0 vs 5 mmol/L). B, T.Tn cells, *P = .88 (0 vs 3 mmol/L), and **P = 0.16 (0 vs 5 mmol/L). A cell invasion assay was carried out. The invading cells were fixed and stained at 48 h after seeding. C, TE2 cells, *P = .23 (0 vs 3 mmol/L), and **P = .04 (0 vs 5 mmol/L). D, T.Tn cells, *P = .18 (0 vs 3 mmol/L), and **P = .13 (0 vs 5 mmol/L)
Figure 6
Figure 6
Western blotting analysis of E‐cadherin protein expression in esophageal squamous cell carcinoma (ESCC) cell lines. A, In TE2 cells, expression of E‐cadherin at each metformin dosage (0, 0.1, 0.3, 1, 3 mmol/L) for 24 h was examined, and the expression level of β‐actin was used as an endogenous control. B, In T.Tn and TE14 cells, expression of E‐cadherin at each metformin dosage (0, 0.3, 1, 3 mmol/L) was evaluated. C, In another several ESCC cell strains, E‐cadherin expression at the control and metformin dosage (3 mmol/L) is shown
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