Sitagliptin affects gastric cancer cells proliferation by suppressing Melanoma-associated antigen-A3 expression through Yes-associated protein inactivation

Abstract

Sitagliptin is an emerging oral hypoglycemic agent that inhibits the development of a wide variety of tumors. Current researches indicate that the abnormal activation of Yes-associated protein (YAP) promotes the proliferation and poor prognosis of multiple tumors. However, the ability of sitagliptin to regulate YAP and its effects on gastric cancer (GC) cells remain unclear. Here, we first showed that sitagliptin inhibited the proliferation of GC cells, and this inhibition was regulated by Hippo pathway. Sitagliptin phosphorylated YAP in a large tumor suppressor homolog-dependent manner, thereby inhibiting YAP nuclear translocation, and promoted YAP cytoplasm retention. This inhibition can be blocked by adenosine 5'-monophosphate-activated protein kinase (AMPK). Moreover, sitagliptin could reduce the expression of tumor-testis antigen Melanoma-associated antigen-A3 through YAP. In conclusion, sitagliptin may have a potential inhibitory effect on GC by AMPK/YAP/melanoma-associated antigen-A3 pathway.

Keywords: MAGE-A3; YAP; gastric cancer; sitagliptin.

FIGURE 1

Sitagliptin inhibits gastric cancer cells proliferation. A, The structure of sitagliptin; (B) The CCK8 assay was used to analyze the inhibition rate after AGS, HGC‐27, and MKN45 cells treated with 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 mmol/L sitagliptin for 24 h; (C) Colony formation assay was performed to assess the long‐term effect of sitagliptin on AGS, HGC‐27, and MKN45 cells; (D) statistically evaluated the difference in clone numbers between treatment groups. NS represented not significant; **P < .01; ***P < .005; ****P < .001

FIGURE 2

Sitagliptin induces Yes‐associated protein (YAP) phosphorylation in gastric cancer cells. A, Immunofluorescence was used to examine the distribution of YAP in sitagliptin‐treated gastric cancer cells. Green represented YAP, blue represented the nucleus (magnification 400×, scale bar 20 µm); (B) the nuclear and cytoplasmic protein extraction technology was used to clarify the effect of sitagliptin on the expression of YAP in the nucleus. The protein expression of YAP was examined using Western blot. GAPDH was used as cytoplasmic reference, while Histon‐H3 was used as nuclear reference; (C) Western blot analysis was used to evaluate p‐large tumor suppressor homolog (LATS) (Ser 909), LATS, p‐YAP (Ser 127), and YAP protein expression with GAPDH as control; (D, E) the peak level of phosphorylation normalized by total protein level compared with control group. Each cell line had a peak of phosphorylation; (F) sitagliptin was added to the gastric cancer (GC) cells, treated with AGS for 6 h, and HGC‐27 and MKN45 for 12 h. YAP and p‐YAP were examined using Western blot; (G) the effects of sitagliptin on the mRNA expression of connective tissue growth factor (CTGF) and cysteine‐rich angiogenic inducer 61 (CYR61). **P < .01; ***P < .005; ****P < .001

FIGURE 3

AMP‐activated protein kinase (AMPK) inhibition reverses sitagliptin‐induced Yes‐associated protein (YAP) phosphorylation in gastric cancer (GC) cells. (A) Western blot analysis was carried out to evaluate the expression of p‐AMPK and AMPK, with GAPDH as control; (B) the peak level of phosphorylation normalized by total protein level compared with control group; (C) after pretreated with or without 10 µm Compound C, GC cells were treated with sitagliptin (AGS for 4 h, HGC‐27 for 12 h and MKN45 for 10 h). Western blot analysis was used to evaluate p‐LATS (Ser 909) and LATS protein expression. GAPDH was used as control; (D) the peak level of phosphorylation normalized by total protein level compared with control group; (E) after pretreated with or without 10 µm Compound C, GC cells were treated with sitagliptin (AGS for 6 h, HGC‐27 for 12 h and MKN45 for 12 h). Western blot analysis was used to evaluate p‐YAP (Ser 127) and YAP protein expression; (F) the peak level of phosphorylation normalized by total protein level compared with control group. *P < .05; **P < .01; ***P < .005; ****P < .001

FIGURE 4

Sitagliptin inhibits melanoma‐associated antigen‐A3 (MAGE‐A3) expression in gastric cancer cells. A and B, The mRNA and protein expression of MAGE‐A3 in gastric cancer (GC) cells after sitagliptin treatment; (C) Statistical evaluation of differences in MAGE‐A3 protein levels; (D) after transfection with Yes‐associated protein (YAP) 5SA or YAP control, the mRNA expression of connective tissue growth factor (CTGF), cysteine‐rich angiogenic inducer 61 (CYR61), and MAGE‐A3 were examined using real‐time PCR; (E) after transfection with YAP 5SA or YAP control, cell viability was tested by CCK8 assay; (F) after transfection with YAP 5SA or YAP control, cells were treated with sitagliptin (1 mmol/L for AGS and HGC‐27; 2 mmol/L for MKN45). MAGE‐A3 protein expression were examined by Western blot, with GAPDH as control; (G) after transfection with siRNA YAP or siRNA control, MAGE‐A3 mRNA expression was confirmed using real‐time PCR; (H) the expression of YAP and MAGE‐A3 were examined to confirm that knocking down YAP did reduce the level of MAGE‐A3; (I, J) statistical evaluation of differences in YAP and MAGE‐A3 protein levels. *P < .05; **P < .01; ***P < .005; ****P < .001

FIGURE 5

Correlation between melanoma‐associated antigen‐A3 (MAGE‐A3) expression and gastric cancer survival. A, MAGE‐A3 expression was determined in gastric cancer (GC) by immunohistochemistry (IHC) (low differentiation; moderate differentiation; high differentiation). The data were shown at different magnification (4 × and 40×); (B) IHC expression of MAGE‐A3 quantified by staining score (0‐12) in GC tissues; (C) Western blot analysis was used to evaluate MAGE‐A3 expression in AGS, HGC‐27, and MKN45 cells, with GAPDH as control; (D, E) knocking down MAGE‐A3 expression in HGC‐27 cells by targeted siRNA transfection. The mRNA and protein expression of MAGE‐A3 were examined; (F) colony formation assay was performed to assess the long‐term effect of siRNA MAGE‐A3 on HGC‐27 cells; (G) statistically evaluated the difference in clone numbers between treatment groups; (H) CCK8 assay was used to assess the cell viabilities of HGC‐27 cells treated with siRNA MAGE‐A3 or siRNA control for different days; (I) the Kaplan‐Meier plotter website data were used for a survival analysis. *P < .05; **P < .01; ***P < .005; ****P < .001

FIGURE 6

Working model. Sitagliptin affects gastric cancer cells proliferation by suppressing melanoma‐associated antigen‐A3 (MAGE‐A3) expression through Yes‐associated protein (YAP) inactivation