TIPE‑2 suppresses growth and aggressiveness of hepatocellular carcinoma cells through downregulation of the phosphoinositide 3‑kinase/AKT signaling pathway

Abstract

Rapid proliferation and migration are the main features of hepatocellular carcinoma (HCC) cells, which serve an essential role in carcinogenesis and are a hallmark of cancer therapy resistance. Previous studies have reported that tumor necrosis factor‑α‑induced protein‑8 like‑2 (TIPE‑2) is involved in cancer initiation and the progression of HCC. The present study aimed to clarify the role of TIPE‑2 in HCC carcinogenesis, growth and aggressiveness. The effects of TIPE‑2 on HCC were determined using colony forming and cell cycle analyses. Cell apoptosis, and growth and aggressiveness of HCC cells, were investigated following TIPE‑2 treatment. Treatment with TIPE‑2 markedly suppressed HCC cell proliferation and increased the number of cells in S phase of the cell cycle. The results demonstrated that TIPE‑2 significantly inhibited growth, migration and invasion of HCC cells via the downregulation of tumor metastasis-associated genes. Flow cytometric analysis indicated that TIPE‑2 promoted apoptosis of HCC cells via regulation of apoptosis‑associated gene transcription. In addition, TIPE‑2 administration downregulated the expression of phosphoinositide 3‑kinase (PI3K) and protein kinase B (AKT) in HCC cells. In addition, TIPE‑2 selectively decreased neuroblastoma Ras viral oncogene and p27 expression in HCC cells. In vivo assays revealed that TIPE‑2 significantly inhibited tumor growth and prolonged animal survival by promoting apoptosis of tumor cells. The results of the present study indicated that TIPE‑2 acts as an inhibitor of HCC cell growth and aggressiveness, and promotes apoptosis, thus suggesting that TIPE‑2 may inhibit the metastasis‑associated PI3K/AKT signaling cascade and may arrest the tumor cell cycle. These findings provide a potential molecular mechanism by which TIPE‑2 promotes apoptosis of HCC cells.

Keywords: tumor necrosis factor-α-induced protein-8 like-2; hepatocellular carcinoma cells; apoptosis; metastasis; phospho-inositide 3-kinase/protein kinase B.

Figure 1.

Inhibitory effects of TIPE-2 on HCC cell growth and proliferation in vitro. (A) TIPE-2 inhibited HepG2 cell growth in a dose- and (B) time-dependent manner. (C) TIPE-2 treatment suppressed HCC cell proliferation and (D) arrested HCC cells at S phase of the cell cycle. (E) Effects of TIPE-2 on the expression levels of cyclin D1 CDK1 and CDK2 in HepG2 cells. (F) Effects of TIPE-2 on the expression levels of Ki67 and PCNA in HepG2 cells, as determined by immunofluorescence. Magnification, ×40. **P<0.01 vs. control group. CDK1, cyclin-dependent kinase; HCC, hepatocellular carcinoma; PCNA, proliferating cell nuclear antigen; TIPE-2, tumor necrosis factor-α-induced protein-8 like-2.

Figure 2.

Inhibitory effects of TIPE-2 (2.0 mg/ml) on hepatocellular carcinoma cell migration and invasion in vitro. Effects of TIPE-2 on (A) migration and (B) invasion of HepG2 cells. Effects of TIPE-2 on the expression levels of (C) VIM, CT-I and Slug, and (D) JNK and NF-κB in HepG2 cells. (E) Immunofluorescence analysis of E-cadherin/DNA and fibronectin/DNA expression levels following TIPE-2 treatment in HepG2 cells. (F) Effects of TIPE-2 on viability of HepG2 cells. Magnification, ×40. **P<0.01 vs. control group. CT-I, collagen type I; JNK, c-Jun N-terminal kinase; NF-κB, nuclear factor-κB; TIPE-2, tumor necrosis factor-α-induced protein-8 like-2; VIM, vimentin.

Figure 3.

Effects of TIPE-2 (2.0 mg/ml) on hepatocellular carcinoma cell apoptosis in vitro. (A) Effects of TIPE-2 on apoptosis of HepG2 cells after 48 h, as determined by flow cytometry. Effects of TIPE-2 treatment on the expression levels of (B) caspase-3 and caspase-8, (C) P53 and Bcl-2, (D) peIF2α and eIF2α, (E) NRF2 and BIP, and (F) GRP78 and CHOP in HepG2 cells, as determined by western blotting. **P<0.01 vs. control group. Bcl-2, B-cell lymphoma 2; BIP, binding immunoglobulin protein; CHOP, CCAAT-enhancer-binding protein homologous protein; eIF2α, eukaryotic initiation factor; FITC, fluorescein isothiocyanate; GRP78, glucose-regulated protein 78; peIF2α, phosphorylated-eIF2α; PI, propidium iodide; TIPE-2, tumor necrosis factor-α-induced protein-8 like-2.

Figure 4.

TIPE-2 regulates hepatocellular carcinoma cell apoptosis through regulation of the PI3K/AKT signaling pathway. Effects of TIPE-2 treatment on the expression levels of (A) PI3K and AKT, (B) N-ras and P27, and (C) pAKT in HepG2 cells. (D) TIPE-2 decreased the ratio of pAkt:Akt in HepG2 cells. Transfection with (E) pedue12.4-PI3K or (F) TIPE-2OR upregulates PI3K or TIPE-2 mRNA expression in HepG2 cells, respectively. **P<0.01 vs. control group. (G) PI3KOR prevented the apoptosis promoted by TIPE-2 treatment in HepG2 cells **P<0.01, PI3KOR-TIPE-2 vs. PI3KOR or control group. Effects of PI3KOR on (H) N-ras and BIP, and (I) P53 and Bcl-2 expression in HepG2 cells. **P<0.01, PI3KOR-TIPE-2 or PI3KOR vs. control group. AKT, protein kinase B; Bcl-2, B-cell lymphoma 2; FITC, fluorescein isothiocyanate; N-ras, neuroblastoma Ras viral oncogene; OR, overexpression; pAKT, phosphorylated-AKT; PI, propidium iodide; PI3K, phosphoinositide 3-kinase; TIPE-2, tumor necrosis factor-α-induced protein-8 like-2.

Figure 4.

TIPE-2 regulates hepatocellular carcinoma cell apoptosis through regulation of the PI3K/AKT signaling pathway. Effects of TIPE-2 treatment on the expression levels of (A) PI3K and AKT, (B) N-ras and P27, and (C) pAKT in HepG2 cells. (D) TIPE-2 decreased the ratio of pAkt:Akt in HepG2 cells. Transfection with (E) pedue12.4-PI3K or (F) TIPE-2OR upregulates PI3K or TIPE-2 mRNA expression in HepG2 cells, respectively. **P<0.01 vs. control group. (G) PI3KOR prevented the apoptosis promoted by TIPE-2 treatment in HepG2 cells **P<0.01, PI3KOR-TIPE-2 vs. PI3KOR or control group. Effects of PI3KOR on (H) N-ras and BIP, and (I) P53 and Bcl-2 expression in HepG2 cells. **P<0.01, PI3KOR-TIPE-2 or PI3KOR vs. control group. AKT, protein kinase B; Bcl-2, B-cell lymphoma 2; FITC, fluorescein isothiocyanate; N-ras, neuroblastoma Ras viral oncogene; OR, overexpression; pAKT, phosphorylated-AKT; PI, propidium iodide; PI3K, phosphoinositide 3-kinase; TIPE-2, tumor necrosis factor-α-induced protein-8 like-2.

Figure 5.

In vivo anticancer effects of TIPE-2 (6.0 mg/kg) treatment on hepatocellular carcinoma-bearing mice. (A) Antitumor effects of TIPE-2 treatment on HepG2-bearing nude mice. (B) TIPE-2 treatment promoted apoptosis of cells within tumor tissues. (C) TIPE-2 treatment increased the expression levels of caspase-3 and caspase-8 in tumor tissues. Effects of TIPE-2 treatment on the expression levels of (D) PI3K and AKT, and (E) GRP78 and CHOP in tumor tissues. Magnification, ×40. (F) Survival rate of HepG2-bearing mice following treatment with TIPE-2 or PBS. **P<0.01 vs. control group. AKT, protein kinase B; CHOP, CCAAT-enhancer-binding protein homologous protein; GRP78, glucose-regulated protein 78; PI3K, phosphoinositide 3-kinase; TIPE-2, tumor necrosis factor-α-induced protein-8 like-2.

Figure 5.

In vivo anticancer effects of TIPE-2 (6.0 mg/kg) treatment on hepatocellular carcinoma-bearing mice. (A) Antitumor effects of TIPE-2 treatment on HepG2-bearing nude mice. (B) TIPE-2 treatment promoted apoptosis of cells within tumor tissues. (C) TIPE-2 treatment increased the expression levels of caspase-3 and caspase-8 in tumor tissues. Effects of TIPE-2 treatment on the expression levels of (D) PI3K and AKT, and (E) GRP78 and CHOP in tumor tissues. Magnification, ×40. (F) Survival rate of HepG2-bearing mice following treatment with TIPE-2 or PBS. **P<0.01 vs. control group. AKT, protein kinase B; CHOP, CCAAT-enhancer-binding protein homologous protein; GRP78, glucose-regulated protein 78; PI3K, phosphoinositide 3-kinase; TIPE-2, tumor necrosis factor-α-induced protein-8 like-2.