Integrin beta-8 (ITGB8) silencing reverses gefitinib resistance of human hepatic cancer HepG2/G cell line

Abstract

Hepatic cancer is a class of cancer that is relatively insensitive to chemotherapy, and cancers that harbor EGFR active mutations are more sensitive to EGFR-TK inhibitor such as gefitinib, which becomes the first-line treatment of this subtype of cancer. However, almost all patients treated with gefitinib will develop drug resistance. Here we show that a protein called integrin beta-8 (ITGB8) when over-expressed, is correlated with the gefitinib resistance of hepatic cancer cell line HepG2/G. After ITGB8 silencing, the drug resistance is reversed as the cell proliferation decreases and apoptosis rate increases significantly by gefitinib treatment when compared to HepG2/G. We demonstrated that multi-drug resistant proteins ABCB1, ABCC2 and ABCG2, anti-apoptosis proteins like survivin and Bcl-2, and cycle promoting protein CDK1 are involved in drug resistance of HepG2/G. Other drug-resistance relative proteins like SOD, GST, TS and HIF-1 are also modulated by ITGB8 silencing, but their role in this gefitinib resistance might be indirect. TGF beta pathway could be a critical pathway by which ITGB8 modulates the sensitivity of HepG2/G to gefitinib.

Keywords: Hepatic cancer; gefitinib resistance; integrin beta-8.

Figure 1

ITGB8 up-regulates in HepG2/G cells and participates in gefitinib resistance. A: Gefitinib IC50 of HepG2 and HepG2/G cells was determined. The data are shown as the means ± SD (n = 3). B: HepG2/G cells were infected with two different ITGB8 shRNAs (shRNA-1 or shRNA-2). Then ITGB8 expression was determined by western blot assay. C: Growth curve of HepG2 cells (Parent), HepG2/G cells (Control) and HepG2/G cells infected with shRNA-1 (shRNA-1) or shRNA-2 (shRNA-2) was determined by MTS assay. D: Cells treated with gefitinib for 72 hours, then cell viability was examined by MTS assay. The data are shown as the means ± SD (n = 3). *P < 0.05, **P < 0.01.

Figure 2

ITGB8 silencing increases accumulation Rhodamine-123 accumulation, cell cycle arrest and apoptosis rate. A and B: Rhodamin-123 accumulation assay was performed on HepG2 cells (Parent), HepG2/G cells (Control) and HepG2/G cells infected with shRNA-1 (shRNA-1) or shRNA-2 (shRNA-2). The data are shown as the means ± SD (n = 3). C and D: HepG2 cells (Parent), HepG2/G cells (Control) and HepG2/G cells infected with shRNA-1 (shRNA-1) or shRNA-2 (shRNA-2) was treated with gefitinib for 24 hours, then Cell cycle phase was analyzed by flow cytometry. The data are shown as the means ± SD (n = 3). E and F: HepG2 cells (Parent), HepG2/G cells (Control) and HepG2/G cells infected with shRNA-1 (shRNA-1) or shRNA-2 (shRNA-2) was treated with gefitinib for 24 hours, then apoptosis rate was determined. The data are shown as the means ± SD. *P < 0.05, **P < 0.01.

Figure 3

ITGB8 silencing induces downregulation of ABCB1, ABCC2 and ABCG2. A and B: qPCR and Western blot analysis of RASAL2 levels in HepG2 cells (Parent), HepG2/G cells (Control) and HepG2/G cells infected with shRNA- 1 (shRNA-1) or shRNA-2 (shRNA-2). The data are shown as the means ± SD. *P < 0.05, **P < 0.01.

Figure 4

ITGB8 silencing down-regulates SOD, GST, TS (Thymidylate synthase), HIF-1 and TGF-beta. Western blot analysis of indicated protein expression in HepG2 cells (Parent), HepG2/G cells (Control) and HepG2/G cells infected with shRNA-1 (shRNA-1) or shRNA-2 (shRNA-2).

Figure 5

ITGB8 modulated the expression of Survivin, Bcl-2, and CDK1. A and B: qPCR and Western blot analysis of Survivin, Bcl-2, and CDK1 levels in HepG2 cells (Parent), HepG2/G cells (Control) and HepG2/G cells infected with shRNA-1 (shRNA-1) or shRNA-2 (shRNA-2). The data are shown as the means ± SD. *P < 0.05, **P < 0.01.

Figure 6

TGF-beta signaling pathway was involved in ITGB8 mediated gefitinib resistance. Western blot analysis of Smad 2/3 and Akt levels in HepG2 cells (Parent), HepG2/G cells (Control) and HepG2/G cells infected with shRNA-1 (shRNA-1) or shRNA-2 (shRNA-2).