AKT-AMPKα-mTOR-dependent HIF-1α Activation is a New Therapeutic Target for Cancer Treatment: A Novel Approach to Repositioning the Antidiabetic Drug Sitagliptin for the Management of Hepatocellular Carcinoma

Abstract

HIF-1α is a key factor promoting the development of hepatocellular carcinoma (HCC). As well, AKT-AMPKα-mTOR signaling is a promising target for cancer therapy. Yet, the AKT-AMPKα-mTOR-dependent activation of HIF-1α has not been studied in livers with HCC. In addition, the mechanisms underlying the potential antineoplastic effects of sitagliptin (STGPT), an antidiabetic agent, have not yet been elucidated. For that purpose, the N-nitrosodiethylamine (NDEA)-induced HCC mouse model was used in the present study using a dose of 100 mg/kg/week, i.p., for 8 weeks. NDEA-induced HCC mice received STGPT 20, 40, or 80 mg/kg starting on day 61 up to day 120. The present study revealed that STGPT inhibited HIF-1α activation via the interference with the AKT-AMPKα-mTOR axis and the interruption of IKKβ, P38α, and ERK1/2 signals as well. Accordingly, STGPT prolonged the survival, restored the histological features and improved liver function. Additionally, STGPT inhibited angiogenesis, as revealed by a significant downregulation in the VEGF and mRNA expression of CD309 with concomitant inhibition of tissue invasion was evident by an increased ratio of TIMP-1/MMP-2. STGPT exhibited apoptotic stimulatory effect as indicated upon calculating the BCL-2/Bax ratio and by the gene expression of p53. The decrease in AFP and liver index calculation, gene expression of Ki-67 confirmed the antiproliferative activity of STGPT. The anti-inflammatory potential was revealed by the decreased TNF-α level and the downregulation of MCP-1 gene expression. Moreover, an antifibrotic potential was supported by lower levels of TGF-β. These effects appear to be GLP1R-independent. The present study provides a potential basis for repurposing STGPT for the inhibition of HCC progression. Since STGPT is unlikely to cause hypoglycemia, it may be promising as monotherapy or adjuvant therapy to treat diabetic or even normoglycemic patients with HCC.

Keywords: AMPKα; Akt; HIF-1α; MAPK; angiogenesis; hepatocellular carcinoma; mTOR; sitagliptin.

FIGURE 1

Effect of STGPT (20, 40, and 80 mg/ kg) on survival rate (A,B,C) in mice with NDEA-induced HCC. Control, normal control group received the vehicle; STGPT 80, normal group received sitagliptin (80 mg/ kg); NDEA, NDEA-induced HCC group received the vehicle; NDEA + STGPT 20, NDEA-induced HCC group treated with sitagliptin (20 mg/kg); NDEA + STGPT 40, NDEA-induced HCC group treated with sitagliptin (40 mg/kg); NDEA + STGPT 80, NDEA-induced HCC group treated with sitagliptin (80 mg/kg). Data in figure (d) are presented as the mean ± SD (n = 6). ⁺⁺⁺⁺ p < 0.0001 vs. Control group, ****p < 0.0001 vs. NDEA group.

FIGURE 2

Effect of STGPT (20, 40, and 80 mg/kg) on histopathological characteristics and necroinflammatory score in mice with NDEA-induced HCC. Representative histological appearance of liver tissue specimens from Control and STGPT 80 showing normal hepatic architecture; Liver sections from NDEA group (left and right panels) showing disrupted hepatic architecture due to the disorganization of hepatic cords, dilatation of hepatic sinusoids (short open arrow) with inflammatory cell infiltration (long open arrows), spotty necrosis (filled arrowhead) and a great number of hepatocytes having large nuclei with prominent and multiple nucleoli (notched arrowhead); Liver sections from NDEA + STGPT 20 and NDEA + STGPT 40 (left panel and right panel, respectively) showing mild disruption of hepatic architecture with dilated sinusoids (short open arrow), decreased intralobular leukocyte infiltration (long open arrow), small number of hepatocytes having large nuclei; Liver sections from NDEA + STGPT 80 showing normal hepatic sinusoids (open arrowhead), decreased disorganization and restored hepatic architecture, very mild intralobular leukocyte infiltration, and diminished hepatocytes having large nuclei. H&E, Bar = as indicated. Calculation of the necroinflammation score reveals that Control and STGPT 80 groups show a score of zero and that treatment groups, particularly the NDEA + STGPT 80, significantly decreased the necroinflammation score compared with that of the NDEA-treated group of rats. Data are presented as the median ± interquartile range (n = 6). ⁺ p < 0.05 vs. Control group, ⁺⁺ p < 0.01 vs. Control group, ⁺⁺⁺ p < 0.001 vs. Control group, ⁺⁺⁺⁺ p < 0.0001 vs. Control group, *p < 0.05 vs. NDEA group, **p < 0.01 vs. NDEA group, ***p < 0.001 vs. NDEA group, ****p < 0.0001 vs NDEA group, ^# p < 0.05 vs STGPT 20 group, ^## p < 0.vs. STGPT 20 group, ^### p < 0.001 vs. STGPT 20 group, ^#### p < 0.0001 vs STGPT 20 group, ^@ p < 0.05 vs. STGPT 40 group, ^@@ p < 0.01 vs. STGPT 40 group, ^@@@ p < 0.001 vs. STGPT 40 group, ^@@@@ p < 0.0001 vs. STGPT 40 group. Control, normal control group received the vehicle; STGPT 80, normal group received sitagliptin (80 mg/kg); NDEA, NDEA-induced HCC group received the vehicle; NDEA + STGPT 20, NDEA-induced HCC group treated with sitagliptin (20 mg/kg); NDEA + STGPT 40, NDEA-induced HCC group treated with sitagliptin (40 mg/kg); NDEA + STGPT 80, NDEA-induced HCC group treated with sitagliptin (80 mg/kg).

FIGURE 3

Effect of STGPT (20, 40 and 80 mg/kg) on ALT (A); AST (B); ALP (C); γ-GT (D); and TAC (E) in mice with NDEA-induced HCC. Data are presented as the mean ± SD (n = 6). ⁺ p < 0.05 vs. Control group, ⁺⁺ p < 0.01 vs. Control group, ⁺⁺⁺ p < 0.001 vs. Control group, ⁺⁺⁺⁺ p < 0.0001 vs. Control group, **p < 0.01 vs NDEA group, ***p < 0.001 vs. NDEA group, ****p < 0.0001 vs. NDEA group, ^### p < 0.001 vs STGPT 20 group, ^@@ p < 0.01 vs STGPT 40 group, ^@@@@ p < 0.0001 vs STGPT 40 group. Control, normal control group received the vehicle; STGPT 80, normal group received sitagliptin (80 mg/kg); NDEA, NDEA-induced HCC group received the vehicle; NDEA + STGPT 20, NDEA-induced HCC group treated with sitagliptin (20 mg/kg); NDEA + STGPT 40, NDEA-induced HCC group treated with sitagliptin (40 mg/kg); NDEA + STGPT 80, NDEA-induced HCC group treated with sitagliptin (80 mg/kg).

FIGURE 4

Effect of STGPT (20, 40 and 80 mg/kg) on TNF-α (A); TGF-β (B); and AFP (C) in mice with NDEA-induced HCC. Data are presented as the mean ± SD (n = 6). ⁺⁺ p < 0.01 vs Control group, ⁺⁺⁺ p < 0.001 vs Control group, ⁺⁺⁺⁺ p < 0.0001 vs Control group, ***p < 0.001 vs NDEA group, ****p < 0.0001 vs NDEA group, ^# p < 0.05 vs STGPT 20 group, ^## p < 0.01 vs STGPT 20 group, ^@ p < 0.05 vs STGPT 40 group, ^@@ p < 0.01 vs STGPT 40 group. Control, normal control group received the vehicle; STGPT 80, normal group received sitagliptin (80 mg/kg); NDEA, NDEA-induced HCC group received the vehicle; NDEA + STGPT 20, NDEA-induced HCC group treated with sitagliptin (20 mg/kg); NDEA + STGPT 40, NDEA-induced HCC group treated with sitagliptin (40 mg/kg); NDEA + STGPT 80, NDEA-induced HCC group treated with sitagliptin (80 mg/kg).

FIGURE 5

Effect of STGPT (20, 40 and 80 mg/kg) on p-AKT (ser473)/AKT (A); and FOXO3 mRNA (B) in mice with NDEA-induced HCC. Data are presented as the mean ± SD (n = 6). ⁺⁺ p < 0.01 vs. Control group, ⁺⁺⁺ p < 0.001 vs. Control group, ⁺⁺⁺⁺ p < 0.vs.vs Control group, ***p < 0.vs. NDEA group, ****p < 0.0001 vs. NDEA group, ^#### p < 0.vs. STGPT 20 group, ^@@@@ p < 0.0001 vs. STGPT 40 group. Control, normal control group received the vehicle; STGPT 80, normal group received sitagliptin (80 mg/kg); NDEA, NDEA-induced HCC group received the vehicle; NDEA + STGPT 20, NDEA-induced HCC group treated with sitagliptin (20 mg/kg); NDEA + STGPT 40, NDEA-induced HCC group treated with sitagliptin (40 mg/kg); NDEA + STGPT 80, NDEA-induced HCC group treated with sitagliptin (80 mg/kg).

FIGURE 6

Effect of STGPT (20, 40 and 80 mg/kg) on p-AMPKα (Ser 487)/AMPKα (A); p-mTOR (S2448) (B); and ULK1 (C) in mice with NDEA-induced HCC. Data are presented as the mean ± SD (n = 6). ⁺ p < 0.05 vs. Control group, ⁺⁺⁺⁺ p < 0.0001 vs. Control group, *p < 0.05 vs NDEA group, **p < 0.01 vs. NDEA group, ****p < 0.0001 vs. NDEA group, ^### p < 0.001 vs. STGPT 20 group, ^#### p < 0.0001 vs. STGPT 20 group, ^@@@ p < 0.001 vs. STGPT 40 group, ^@@@@ p < 0.0001 vs. STGPT 40 group. Control, normal control group received the vehicle; STGPT 80, normal group received sitagliptin (80 mg/kg); NDEA, NDEA-induced HCC group received the vehicle; NDEA + STGPT 20, NDEA-induced HCC group treated with sitagliptin (20 mg/kg); NDEA + STGPT 40, NDEA-induced HCC group treated with sitagliptin (40 mg/kg); NDEA + STGPT 80, NDEA-induced HCC group treated with sitagliptin (80 mg/kg).

FIGURE 7

Effect of STGPT (20, 40 and 80 mg/kg) on p-p38 MAPKα (A); p-Ikkβ (B); and ERK1/2 (C) in mice with NDEA-induced HCC. Data are presented as the mean ± SD (n = 6). ⁺⁺⁺ p < 0.001 vs. Control group, ⁺⁺⁺⁺ p < 0.0001 vs. Control group, ***p < 0.001 vs. NDEA group, ****p < 0.0001 vs. NDEA group, ^#### p < 0.0001 vs. STGPT 20 group, ^@@ p < 0.01 vs. STGPT 40 group, ^@@@@ p < 0.0001 vs. STGPT 40 group. Control, normal control group received the vehicle; STGPT 80, normal group received sitagliptin (80 mg/kg); NDEA, NDEA-induced HCC group received the vehicle; NDEA + STGPT 20, NDEA-induced HCC group treated with sitagliptin (20 mg/kg); NDEA + STGPT 40, NDEA-induced HCC group treated with sitagliptin (40 mg/kg); NDEA + STGPT 80, NDEA-induced HCC group treated with sitagliptin (80 mg/kg).

FIGURE 8

Effect of STGPT (20, 40 and 80 mg/kg) on BCL-2 (A); Bax (B); BCL-2: Bax ratio (C); p53 mRNA (D); and Ki-67 mRNA (E) in mice with NDEA-induced HCC. Data are presented as the mean ± SD (n = 6). ⁺ p < 0.05 vs Control group, ⁺⁺ p < 0.01 vs. Control group, ⁺⁺⁺ p < 0.001 vs. Control group, ⁺⁺⁺⁺ p < 0.0001 vs. Control group, *p < 0.05 vs. NDEA group, **p < 0.01 vs. NDEA group, ***p < 0.001 vs. NDEA group, ****p < 0.0001 vs. NDEA group, ^#### p < 0.0001 vs. STGPT 20 group, ^@@ p < 0.01 vs. STGPT 40 group, ^@@@@ p < 0.0001 vs. STGPT 40 group. Control, normal control group received the vehicle; STGPT 80, normal group received sitagliptin (80 mg/kg); NDEA, NDEA-induced HCC group received the vehicle; NDEA + STGPT 20, NDEA-induced HCC group treated with sitagliptin (20 mg/kg); NDEA + STGPT 40, NDEA-induced HCC group treated with sitagliptin (40 mg/kg); NDEA + STGPT 80, NDEA-induced HCC group treated with sitagliptin (80 mg/kg).

FIGURE 9

Effect of STGPT (20, 40 and 80 mg/kg) on TIMP-1/MMP-2 ratio (A); VEGF (B); CD309 mRNA (C); and HIF1-α (D) in mice with NDEA-induced HCC. Data are presented as the mean ± SD (n = 6). ⁺ p < 0.05 vs. Control group, ⁺⁺ p < 0.01 vs. Control group, ⁺⁺⁺ p < 0.001 vs Control group, ⁺⁺⁺⁺ p < 0.0001 vs Control group, **p < 0.01 vs NDEA group, ***p < 0.001 vs. NDEA group, ****p < 0.0001 vs. NDEA group, ^## p < 0.01 vs STGPT 20 group, ^#### p < 0.0001 vs. STGPT 20 group, ^@ p < 0.05 vs. STGPT 40 group. Control, normal control group received the vehicle; STGPT 80, normal group received sitagliptin (80 mg/kg); NDEA, NDEA-induced HCC group received the vehicle; NDEA + STGPT 20, NDEA-induced HCC group treated with sitagliptin (20 mg/kg); NDEA + STGPT 40, NDEA-induced HCC group treated with sitagliptin (40 mg/kg); NDEA + STGPT 80, NDEA-induced HCC group treated with sitagliptin (80 mg/kg).

FIGURE 10

Effect of STGPT (20, 40 and 80 mg/kg) on MCP-1 mRNA (A) and GLP-1R mRNA (B) in mice with NDEA-induced HCC. Data are presented as the mean ± SD (n = 6). ⁺ p < 0.05 vs. Control group, ⁺⁺⁺⁺ p < 0.0001 vs. Control group, **p < 0.01 vs. NDEA group, ****p < 0.0001 vs. NDEA group, ^#### p < 0.0001 vs. STGPT 20 group, ^@@@@ p < 0.0001 vs. STGPT 40 group. Control, normal control group received the vehicle; STGPT 80, normal group received sitagliptin (80 mg/kg); NDEA, NDEA-induced HCC group received the vehicle; NDEA + STGPT 20, NDEA-induced HCC group treated with sitagliptin (20 mg/kg); NDEA + STGPT 40, NDEA-induced HCC group treated with sitagliptin (40 mg/kg); NDEA + STGPT 80, NDEA-induced HCC group treated with sitagliptin (80 mg/kg).