Differential in vitro effects of targeted therapeutics in primary human liver cancer: importance for combined liver cancer

Abstract

The incidence of primary liver tumors, hepatocellular carcinoma (HCC), intrahepatic cholangiocellular carcinoma (ICC), and combined HCC/ICC (cHCC/CC) is increasing. For ICC, targeted therapy exists only for a small subpopulation of patients, while for HCC, Sorafenib and Lenvatinib are in use. Diagnosis of cHCC/CC is a great challenge and its incidence is underestimated, bearing the risk of unintended non-treatment of ICC. Here, we investigated effects of targeted inhibitors on human ICC cell lines (HUH28, RBE, SSP25), in comparison to extrahepatic (E)CC lines (EGI1, CCC5, TFK1), and HCC/hepatoblastoma cell lines (HEP3B, HUH7, HEPG2). Cells were challenged with: AKT inhibitor MK-2206; multikinase inhibitors Sorafenib, Lenvatinib and Dasatinib; PI3-kinase inhibitors BKM-120, Wortmannin, LY294002, and CAL-101; and mTOR inhibitor Rapamycin. Dosage of the substances was based on the large number of published data of recent years. Proliferation was analyzed daily for four days. All cell lines were highly responsive to MK-2206. Thereby, MK-2206 reduced expression of phospho(p)-AKT in all ICC, ECC, and HCC lines, which mostly corresponded to reduction of p-mTOR, whereas p-ERK1/2 was upregulated in many cases. Lenvatinib showed inhibitory effects on the two HCC cell lines, but not on HEPG2, ICCs and ECCs. Sorafenib inhibited proliferation of all cells, except the ECC line CCC5. However, at reduced dosage, we observed increased cell numbers in some ICC experiments. Dasatinib was highly effective especially in ICC cell lines. Inhibitory effects were observed with all four PI3-kinase inhibitors. However, cell type-specific differences were also evident here. Rapamycin was most effective in the two HCC cell lines. Our studies show that the nine inhibitors differentially target ICC, ECC, and HCC/hepatoblastoma lines. Caution should be taken with Lenvatinib and Sorafenib administration in patients with cHCC/CC as the drugs may have no effects on, or might even stimulate, ICC.

Keywords: AKT pathway; Cholangiocellular carcinoma; Combined liver cancer; Hepatocellular carcinoma; Kinase inhibitors.

Fig. 1

Proliferation studies and Western-blot analysis of ICC cell lines. A Effects of AKT inhibitor MK-2206 on HUH28 (a), RBE (b), and SSP25 (c) after 0, 24, 48, 72 and 96 h (h). 0 h values were set to 100%. Concentrations of MK-2206 are indicated. Shown are the mean values of n = 3–5 independent experiments with each 6–8 replicates. Controls were treated with solvent (DMSO). Significance was tested by one-way ANOVA between the control and treatment groups. *p < 0.05, **p < 0.01, ***p < 0.001 with 95% level of confidence. B Cropped Western blot images of 25 µM MK-2206-treated HUH28 and RBE and SSP25 with specific antibodies against total AKT and p-AKT, mTOR and p-mTOR, ERK1/2 and p-ERK1/2 after 3, 8 and 24 h. The samples were derived from the same experiment and the blots were processed parallelly. α-Tubulin was used as loading control. Note reduced phosphorylation of AKT and mTOR and increase in p-ERK1/2, in addition to death of HUH28 cells after 8 h and 24 h of MK-2206

Fig. 2

Proliferation studies and Western-blot analysis of ECC cell lines. A Effects of AKT inhibitor MK-2206 on EGI1 (a), CCC5 (b), and TFK1 (c) after 0, 24, 48, 72 and 96 h (h). 0 h values were set to 100%. Concentrations of MK-2206 are indicated. Shown are the mean values of n = 3–5 independent experiments with each 6–8 replicates. Controls were treated with solvent (DMSO). Significance was tested by one-way ANOVA between the control and treatment groups. *p < 0.05, **p < 0.01, ***p < 0.001 with 95% level of confidence. B Cropped Western blot images of 25 µM MK-2206-treated EGI1 and CCC5 with specific antibodies against total AKT and p-AKT, mTOR and p-mTOR, ERK1/2 and p-ERK1/2 after 3, 8 and 24 h. α-Tubulin was used as loading control. Note reduced phosphorylation of AKT, and increase in p-ERK1/2

Fig. 3

Proliferation studies and Western-blot analysis of HCC/hepatoblastoma cell lines. A Effects of AKT inhibitor MK-2206 on HEP3B (a), HUH7 (b), and HEPG2 (c) after 0, 24, 48, 72 and 96 h (h). 0 h values were set to 100%. Concentrations of MK-2206 are indicated. Shown are the mean values of n = 3–5 independent experiments with each 6–8 replicates. Controls were treated with solvent (DMSO). Significance was tested by one-way ANOVA between the control and treatment groups. *p < 0.05, **p < 0.01, ***p < 0.001 with 95% level of confidence. B Cropped Western blot images of 25 µM MK-2206-treated HEP3B and HUH7 with specific antibodies against total AKT and p-AKT, mTOR and p-mTOR, ERK1/2 and p-ERK1/2 after 3, 8 and 24 h. The samples were derived from the same experiment and the blots were processed parallelly. α-Tubulin was used as loading control. Note reduced phosphorylation of AKT and mTOR, and heterogenous results with p-ERK1/2

Fig. 4

Proliferation studies of ICC (HUH28, RBE, SSP25, a-c), ECC (EGI1, CCC5, TFK1, d-f) and HCC/hepatoblastoma (HEP3B, HUH7, HEPG2, g-i) cell lines. Effects of (A) Sorafenib, (B) Lenvatinib, and (C) Dasatinib after 0, 24, 48, 72 and 96 h (h). 0 h values were set to 100%. Concentrations of Sorafenib are indicated. Shown are the mean values of n = 3–5 independent experiments with each 6–8 replicates. Controls were treated with solvent (DMSO). Significance was tested by one-way ANOVA between the control and treatment groups. *p < 0.05, **p < 0.01, ***p < 0.001 with 95% level of confidence

Fig. 5

Proliferation studies of ICC (HUH28, RBE, SSP25, a-c), ECC (EGI1, CCC5, TFK1, d-f) and HCC/hepatoblastoma (HEP3B, HUH7, HEPG2, g-i) cell lines. Effects of (A) BKM120, (B) Wortmannin (Wort), (C) LY294002 (LY29), and (D) CAL-101 after 0, 24, 48, 72 and 96 h (h). 0 h values were set to 100%. Concentrations of BKM120 are indicated. Shown are the mean values of n = 3–5 independent experiments with each 6–8 replicates. Controls were treated with solvent (DMSO). Significance was tested by one-way ANOVA between the control and treatment groups. *p < 0.05, **p < 0.01, ***p < 0.001 with 95% level of confidence

Fig. 6

Proliferation studies of ICC (HUH28, RBE, SSP25, a-c, ECC (EGI1, CCC5, TFK1, d-f and HCC/hepatoblastoma (HEP3B, HUH7, HEPG2, g-i cell lines. Effects of Rapamycin (Rapa) after 0, 24, 48, 72 and 96 h (h). 0 h values were set to 100%. Concentrations of Rapamycin are indicated. Shown are the mean values of n = 3–5 independent experiments with each 6–8 replicates. Controls were treated with solvent (DMSO). Significance was tested by one-way ANOVA between the control and treatment groups. *p < 0.05, **p < 0.01, ***p < 0.001 with 95% level of confidence