Metformin inhibits proliferation and growth hormone secretion of GH3 pituitary adenoma cells

Abstract

Metformin is an anti-hyperglycemic agent used to treat diabetes, and recent evidence suggests it has antitumor efficacy. Because growth hormone-secreting pituitary adenoma (GH-PA) patients have a high incidence of diabetes frequently treated with metformin, we assessed the antitumor effect of metformin on GH-PA. We found that metformin effectively inhibited proliferation and induced apoptosis in the GH-PA cell line GH3. We detected a decrease in mitochondrial membrane potential (MMP), an increase in expression of pro-apoptotic proteins, and a decrease in expression of an anti-apoptotic protein in metformin-treated GH3 cells, which suggests involvement of the mitochondrial-mediated apoptosis pathway. Inhibition of AMPK, which is activated by metformin, failed to reverse the antiproliferative effect. ATF3 was upregulated by metformin, and its knockdown significantly reduced metformin-induced apoptosis. In addition, GH secretion was inhibited by metformin through suppression of STAT3 activity independently of AMPK. Metformin also significantly suppressed cellular proliferation and GH secretion in primary human GH-PA cells. Metformin also significantly inhibited GH3 cell proliferation and GH secretion in vivo. ATF3 upregulation and p-STAT3 downregulation were confirmed in xenografts. These findings suggest metformin is a potentially promising therapeutic agent for the treatment of GH-PA, particularly in patients with diabetes.

Keywords: ATF3; STAT3; growth hormone-secreting pituitary adenoma; metformin; proliferation.

Figure 1. Metformin (Met) induced apoptosis via the mitochondrial apoptotic pathway in a dose dependent manner

(A) GH3 cells were incubated with different concentrations of metformin for 0, 24, 48, and 72 h, and cell proliferation was measured by CCK-8 assay. (B) GH3 cells were exposed to 0, 2, and 5 mM metformin for 48 h, and then cell cycle was analyzed using flow cytometry. (C) Apoptosis of cells treated with metformin at different concentrations was measured by flow cytometry. (D) Mitochondrial membrane potential (MMP) was measured in different treatment groups by JC-1 staining. (E) Protein expression levels of Bcl2, Bax and Cleaved caspase3 were examined by Western blot. *P < 0.05;**P < 0.01.

Figure 2. Metformin induced GH3 cell apoptosis by activation of ATF3 independent of AMPK

(A) Protein levels of p-AMPK (Thr172) and AMPK were examined by Western blot. (B) GH3 cell proliferation with metformin treatment (5mM for 48 h) was assayed by CCK-8, following treatment with compound C (5 uM). (C and D) ATF3 mRNA and protein levels in 2 groups of treatment with or without metformin for 48 h by RT-qPCR and Western blot. (E and F) ATF3 mRNA and protein levels of cells transfected with control or ATF3 siRNA. (G) GH3 cells were transfected with control or ATF3 siRNA and then treated with 5 mM metformin for 48 h. Cleaved-caspase3 was examined by Western blot. *P < 0.05;**P < 0.01.

Figure 3. Metformin inhibited GH secretion by GH3 cells

(A) GH3 cells were incubated with different concentrations of metformin for 0, 12, 24, 36, 48, 60 and 72 h, and GH secretion was detected by ELISA. (B) GH3 cells were pretreated with or without compound C and then treated with metformin. p-AMPK(Thr172) and p-STAT3(Tyr705) were examined by Western blot. *P < 0.05; **P < 0.01.

Figure 4. Metformin inhibited GH3 cell growth and GH secretion in vivo

(A) GH3 cells were injected subcutaneously in nude mice and excised tumors from different groups are shown. (B) The weight of the excised tumors. (C) Growth curve shows the tumor volume change of mice after administration of vehicle or metformin. (D) Protein level of ATF3 in tumor tissues was examined by Western blot. (E) GH level of mice after different treatments was detected by ELISA. (F) The weight of the mice after different treatments was recorded. (G) Protein level of p-STAT3(Tyr705) in tumor tissues was examined by Western blot. *P < 0.05;**P < 0.01.

Figure 5. Metformin suppressed cellular proliferation and reduced GH secretion in primary cultures of human GH-PA cells

(A) primary GH-PA cells were identified by human GH. Blue signal, DAPI nuclear staining; green signal, GH staining. Original magnification, ×400. (B) The effects of metformin on proliferation of the primary tumor cells, after 72 hours of exposure. (C) Detection of the primary cells GH secretion after treated with metformin by ELISA. *P < 0.05;**P < 0.01.