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. 2014 Feb 27;5(2):e1088.
doi: 10.1038/cddis.2014.59.

Metformin promotes autophagy and apoptosis in esophageal squamous cell carcinoma by downregulating Stat3 signaling

Y Feng  1 , C Ke  2 , Q Tang  1 , H Dong  2 , X Zheng  2 , W Lin  2 , J Ke  1 , J Huang  3 , S-C J Yeung  4 , H Zhang  5
Affiliations

Metformin promotes autophagy and apoptosis in esophageal squamous cell carcinoma by downregulating Stat3 signaling

Y Feng et al. Cell Death Dis. .

Abstract

The antidiabetic drug metformin exerts chemopreventive and antineoplastic effects in many types of malignancies. However, the mechanisms responsible for metformin actions appear diverse and may differ in different types of cancer. Understanding the molecular and cellular mechanisms specific for different cancers is important to optimize strategy for metformin treatment in different cancer types. Here, we investigate the in vitro and in vivo effects of metformin on esophageal squamous cell carcinoma (ESCC) cells. Metformin selectively inhibited cell growth in ESCC tumor cells but not immortalized noncancerous esophageal epithelial cells. In addition to apoptosis, metformin triggered autophagy. Pharmacological or genetic inhibition of autophagy sensitized ESCC cells to metformin-induced apoptotic cell death. Mechanistically, signal transducer and activator of transcription 3 (Stat3) and its downstream target Bcl-2 was inactivated by metformin treatment. Accordingly, small interfering RNA (siRNA)-mediated Stat3 knockdown enhanced metformin-induced autophagy and apoptosis, and concomitantly enhanced the inhibitory effect of metformin on cell viability. Similarly, the Bcl-2 proto-oncogene, an inhibitor of both apoptosis and autophagy, was repressed by metformin. Ectopic expression of Bcl-2 protected cells from metformin-mediated autophagy and apoptosis. In vivo, metformin downregulated Stat3 activity and Bcl-2 expression, induced apoptosis and autophagy, and inhibited tumor growth. Together, inactivation of Stat3-Bcl-2 pathway contributes to metformin-induced growth inhibition of ESCC by facilitating crosstalk between apoptosis and autophagy.

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Figures

Figure 1
Figure 1
Metformin selectively inhibits ESCC cell growth. EC109 (a), EC9706 (b) and NE3 (c) cells were treated with metformin at the indicated concentration for 24 and 48 h and for 7 days. Cell viability, measured by MTT, was presented as the means±SD from three separate experiments. (d) Colony formation of ESCC cells was decreased in a dose-dependent manner by metformin treatment. Experiments were performed in triplicate
Figure 2
Figure 2
Metformin induces apoptotic cell death in ESCC cells. (a) Cells were stained with annexin-V-FITC (20 μg/ml) and PI (20 mg/l) following treatment with or without metformin for 48 and 72 h and for 7 days. Apoptosis was analyzed using flow cytometry. (b) Cells were stained with JC-1 (10 μg/ml) following treatment with or without metformin at the indicated concentrations for 48 h. Mitochondrial membrane potential (MMP) was analyzed using flow cytometry. (c) TUNEL staining of EC109 cells after 48 h of metformin treatment at the indicated concentrations. Positive cells were labeled with TUNEL (green) (magnification, × 400). Quantification of the number of TUNEL-positive cells in the control and metformin-treated cells (graph on the right), plotted as a percentage TUNEL-positive cells. *P<0.05 and **P<0.01 represent significant differences compared with the control. The P-values were calculated using a two-sided Student's t-test. (d) Effects of metformin on apoptosis-related proteins assayed by western blot at different treatment concentrations (left) and different times (right). β-Actin was probed as the loading control. (e) Cell proliferation assessed by immunohistochemistry with anti-phospho-Histone H3 (pH3) antibody in ESCC cells after treatment with metformin for 48 and 72 h and for 7 days. pH3 were red and nuclei were stained with DAPI (blue). Per field, 100 cells were counted, and 10 fields were counted per sample (magnification, × 200). Graphs represent the percentage of pH3-positive cells. **P<0.01 and ***P<0.001 compared with the control. P-values were calculated using a two-sided Student's t-test. Representative data from one of three independent experiments are shown in panels from (a) through (d)
Figure 3
Figure 3
Metformin triggers ESCC cell autophagy. (a) Representative images of AO (upper) and MDC (middle) staining of ESCC cells following treatment with metformin at the indicated concentration for the indicated time. For AO staining (upper), red color intensity shows acidic vesicular organelles, representing autophagolysosomes. For MDC staining (middle), punctate fluorescence in the cytoplasm indicates formation of autophagic vacuoles. In the bottom panel, EC109 cells transfected with GFP-LC3 plasmid were treated with metformin for 24 and 48 h. Control cells showed a diffuse expression pattern of GFP-LC3, whereas metformin-treated cells displayed GFP-LC3-II a punctate pattern, indicating formation of autophagosomes. Magnification: × 400 for AO and MDC stainings, and × 1000 for GFP-LC3. (b) EC109 cells were treated with the indicated concentration of metformin for 48 h, and then cells were stained with AO. Cell autophagy was analyzed by quantification of acidic vesicular organelles (AVOs) with AO using flow cytometry. (c) Effects of metformin on autophagy-related proteins assayed by western blot at different metformin concentrations (left) and different times (right). β-Actin was probed as the loading control. (d) Transmission electron microscopy (TEM) images showing autophagic vacuoles (arrows) observed in metformin-treated EC109 cells for 48 h (right). Starved cells were used as a positive control for autophagy (middle). No or few autophagic vacuoles are observed in control cells (left panel). Representative data from one of three independent experiments are shown in (a–c)
Figure 4
Figure 4
Inhibition of autophagy sensitizes cells to metformin-induced apoptotic cell death. (a) Inhibition of autophagy with 3-MA or CQ decreases viable metformin-treated cells. Cell viability was measured by MTT assay after cells were incubated with the indicated concentration of metformin with or without 3-MA (±3-MA, 5 mM; ±CQ, 25 μM) for 48 h (left and middle). Cell viability was measured by MTT assay after cells were incubated with the indicated concentration of 3-MA with or without metformin (±metformin, 10 mM) for 48 h (right). Data were expressed as the means±SD from three separate experiments as the percentage of viable cells in the control group. *P<0.05 and **P<0.01 by Student's t-test. (b) Representative images of TUNEL staining of EC109 cells after treatment with the indicated concentration of metformin with or without 3-MA (±3-MA) for 48 h (magnification, × 400). Quantification of the number of TUNEL-positive cells (graph on the right), plotted as a percentage TUNEL-positive cells. **P<0.01 by Student's t-test. (c) Cells were stained with JC-1 (10 μg/ml) following metformin treatment with or without metformin CQ (±CQ) for 48 h. Mitochondrial membrane potential (MMP) was analyzed using flow cytometry. (d) Apoptosis- and autophagy-related proteins were examined by western blot following metformin treatment with or without 3-MA (±3-MA) for 48 h. (e) Effects of genetic inhibition of autophagy by knockdown of Beclin-1 or Atg5 on metformin-mediated apoptosis. LC3 and cleaved PARP were examined by western blot. Representative data from one of three independent experiments are shown in (b–e). Error bars indicate±S.E. *P<0.05 and **P<0.01 by Student's t-test. β-Actin was the loading control in (d) and (e)
Figure 5
Figure 5
Inactivation of Stat3/Bcl-2 signaling contributes to metformin-induced effects. (a) Metformin treatment repressed expression of p-Stat3 and cyclin D1, as assayed by western blot. (b) Western blotting was used to assess p-Stat3, Stat3 targets (cyclin D1 and Bcl-2), apoptosis-related proteins, and autophagy-related proteins in Stat3 siRNA – or control siRNA-transfected EC109 cells treated with metformin. (c) Representative images ( × 1000) of GFP-LC3 expression patterns in Stat3 siRNA and control siRNA cells following metformin treatment. EC109 cells transfected with GFP-LC3 plasmid were used. (d) Ectopic expression of Bcl-2 attenuates metformin-mediated autophagy and apoptosis, as indicated by related protein expression assayed by western blot. (e) Representative images of IHC staining of p-Stat3 and Bcl-2 in adjacent nontumor ( × 200) and ESCC tumor tissues ( × 400) from patients. The p-Stat3 protein level positively correlated with the Bcl-2 protein level in ESCC tumors from 36 patient samples (Pearson's correlation coefficient test, r=0.695 and P<0.001). (f) Effects of metformin treatment (10 mM for 48 h) on expression of AMPK/mTOR, p-Stat3 and LC3, as assayed by western blot, following transfection with AMPK siRNA or control siRNA. Representative data from one of three independent experiments are shown in (a, b, c, d and f). β-Actin was applied as the loading control for (a, b, d and f)
Figure 6
Figure 6
Metformin inhibits ESCC tumor growth in vivo. (a) Tumor growth curve of metformin-treated mice (250 mg/kg body weight) and control (vehicle treated) mice. Data represent the mean±S.D. of eight mice in each group. *P<0.05 by the Student's t-test. (b) Dissected tumor was photographed and weight was measured. *P<0.05 by Student's t-test. (c) Representative images ( × 400) of IHC analysis of p-Stat3, cyclin D1, Bcl-2, PCNA, TUNEL and p62 in tumors. (d) Western blots of LC3 in tumor tissues. β-Actin was probed as the loading control
Figure 7
Figure 7
Proposed mechanisms responding to metformin-induced effects in ESCC
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