ARQ-197 enhances the antitumor effect of sorafenib in hepatocellular carcinoma cells via decelerating its intracellular clearance

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the heaviest malignant burdens in China. Molecular targeting agent, sorafenib, is the main therapeutic option for antitumor therapy of advanced HCC, but it is currently too expensive for the public and its therapeutic effect does not satisfy initial expectation. Therefore, it is important to develop more effective molecular targeted therapeutic strategies for advanced HCC.

Materials and methods: The antitumor effects of sorafenib or ARQ-197, an antagonist of c-MET (tyrosine-protein kinase Met or hepatocyte growth factor receptor), were examined by MTT or in murine tumor model. The effect of ARQ-197 on epithelial-mesenchymal transition (EMT) or multidrug resistance (MDR) was examined by quantitative real-time PCR for the expression of related genes. The clearance of sorafenib in HCC cells was detected by liquid chromatography-mass spectrometry/mass spectrometry.

Results: ARQ-197 treatment enhanced the sensitivity of HCC cells to sorafenib. Mechanistic studies indicated that ARQ-197 inhibited the expression of EMT- and MDR-related genes. Moreover, ARQ-197 treatment decelerated the clearance of sorafenib in cultured HCC cells and subcutaneous HCC tumors in nude mice.

Conclusion: In the present work, our data suggested that ARQ-197 decelerated the clearance of sorafenib in HCC cells and enhanced the antitumor effect of sorafenib.

Keywords: ARQ-197 and sorafenib; advanced hepatocellular carcinoma; drug clearance; epithelial–mesenchymal transition; molecular targeted agents; multidrug resistance.

Figure 1

The mRNA level of c-MET in hepatic cells. Notes: Hepatic cells – L-02, HepG2, LM-3, MHCC97-H, Hu7, BEL-7402, SMMC-7721, or MHCC97-L – were harvested for qPCR experiments. Abbreviation: qPCR, quantitative real-time PCR.

Figure 2

ARQ-197 inhibits the survival of HCC cells in a dose-dependent manner. Notes: HCC cells MHCC97-H (A), LM-3 (B), and HepG2 (C), which were treated with indicated concentrations of ARQ-197, were analyzed by MTT experiments. Inhibition rate of ARQ-197 on HCC cells was calculated at OD 490 nm. Abbreviation: HCC, hepatocellular carcinoma.

Figure 3

ARQ-197 enhances the antitumor effect of sorafenib or inhibits the survival of HCC cells in a dose-dependent manner. Notes: The HCC cells MHCC97-H (A), LM-3 (B), and HepG2 (C), which were pretreated with IC₂₅ concentration of ARQ-197, were treated with indicated concentrations of sorafenib. Next, the cells were analyzed by MTT experiments. Inhibition rates of sorafenib on HCC cells were calculated at OD 490 nm. Abbreviation: HCC, hepatocellular carcinoma.

Figure 4

ARQ-197 inhibits EMT- or MDR-related genes’ expression in MHCC97-H cells. Notes: MHCC97-H cells, which were treated with IC₂₅ concentration of ARQ-197, were harvested for qPCR experiments. The mRNA level of EMT-related genes, E-cadherin (A), N-cadherin (B), vimentin (C), or MDR-related genes, CYP3A4 (D), MDR-1 (E), UTG1A9 (F), was examined by qPCR. ^*P<0.05. Abbreviations: EMT, epithelial–mesenchymal transition; MDR, multidrug resistance; qPCR, quantitative real-time PCR.

Figure 5

ARQ-197 inhibits the protein level of EMT- or MDR-related genes’ expression in MHCC97-H cells. Notes: MHCC97-H cells, which were treated with IC₂₅ concentration of ARQ-197, were harvested for Western blot experiments. The protein levels of EMT-related genes, E-cadherin, N-cadherin, vimentin, or MDR-related genes, CYP3A4, MDR-1, UTG1A9, were examined by their antibodies. Abbreviations: EMT, epithelial–mesenchymal transition; MDR, multidrug resistance.

Figure 6

ARQ-197 decreases the transcription factor activation of ETS-1 and PXR in MHCC97-H cells. Notes: MHCC97-H cells which were transfected with luciferase reporters of (A) ETS-1 (EBS-Luc) or luciferase reporters of PXR (DR3-Luc [B] or ER6-Luc [C]) were treated with indicated concentration of ARQ-197. ^*P<0.05. Abbreviations: DR3, direct repeat 3; ER6, everted repeat 6.

Figure 7

ARQ-197 decelerates the clearance of sorafenib in MHCC97-H cells. Notes: (A) MHCC97-H cells, which were treated with IC₂₅ concentration sorafenib for 12 hours, were harvested at indicated time points. (B) Sorafenib solution was injected into subcutaneous tumor formed by MHCC97-H cells, and tumor tissues were harvested at indicated time points. Samples were analyzed by LC-MS/MS. Drugs clearance curve was calculated based on the sustaining of sorafenib in cells or tumors. ^*P<0.05. Abbreviation: LC-MS/MS, liquid chromatography–mass spectrometry/mass spectrometry.

Figure 8

ARQ-197 enhances the antitumor effect of sorafenib on the subcutaneous growth of MHCC97-H cells in nude mice. Notes: MHCC97-H cells were seeded into nude mice to form subcutaneous tumors. Nude mice received oral administration of solvent control, ARQ-197, sorafenib, or ARQ-197 + sorafenib. Tumors were harvested and tumor volumes or tumor weights were noted. Results are shown as photographs of subcutaneous tumors (A), tumor volumes (B), inhibition rates of agents on MHCC97-H cells’ subcutaneous growth calculated from tumor volumes (C), or tumor weights (D), orinhibition rates of agents on MHCC97-H cells’ subcutaneous growth calculated from tumor weights (E). ^*P<0.05.

Figure 8

ARQ-197 enhances the antitumor effect of sorafenib on the subcutaneous growth of MHCC97-H cells in nude mice. Notes: MHCC97-H cells were seeded into nude mice to form subcutaneous tumors. Nude mice received oral administration of solvent control, ARQ-197, sorafenib, or ARQ-197 + sorafenib. Tumors were harvested and tumor volumes or tumor weights were noted. Results are shown as photographs of subcutaneous tumors (A), tumor volumes (B), inhibition rates of agents on MHCC97-H cells’ subcutaneous growth calculated from tumor volumes (C), or tumor weights (D), orinhibition rates of agents on MHCC97-H cells’ subcutaneous growth calculated from tumor weights (E). ^*P<0.05.