Tumor-treating fields in combination with sorafenib restrain the proliferation of liver cancer in vitro

Abstract

Liver cancer is a common malignancy worldwide, with a poor prognosis and a high recurrence rate despite the available treatment methodologies. Tumor-treating fields (TTFields) have shown good preclinical and clinical results for improving the prognosis of patients with glioblastoma and malignant pleural mesothelioma. However, there is minimal evidence for the effect of TTFields on other cancer types. Thus, the present study aimed to investigate the therapeutic efficacy of TTFields in an in vitro model, and to further elucidate the underlying mechanisms. In the present study, two hepatocellular carcinoma (HCC) cell lines (Hep3B and HepG2) were treated with TTFields (intensity, 1.0 V/cm; frequency, 150 kHz) in order to determine the potential antitumor effects of this approach. TTFields significantly inhibited the proliferation and viability of HCC cell lines, as measured using Trypan blue and MTT assays, as well as colony formation in three-dimensional cultures. The TTFields also significantly inhibited the migration and invasion of HCC cells in Transwell chamber and wound-healing assays. Moreover, TTFields enhanced the production of reactive oxygen species in the cells and increased the proportion of apoptotic cells, as evidenced by increased caspase-3 activity, as well as PARP cleavage in western blotting experiments. All of these effects were increased following the application of TTFields in combination with the multi-kinase inhibitor sorafenib, which demonstrated a synergistic effect. Thus, to the best of our knowledge, these results demonstrate for the first time the potential of TTFields in improving the sensitivity of HCC cells to sorafenib, which may lay the foundation for future clinical trials for this combination treatment strategy.

Keywords: apoptosis; liver cancer; sorafenib; tumor-treating fields.

Figure 1.

Effect of TTFields on the viability of liver cancer cells. (A) Experimental scheme of treatment with TTFields in liver cancer cell lines. (B) The analysis of liver cancer cell viability analysis according to the frequency and the intensity. (C) The viability of cells treated with TTFields was significantly lower than that of control cells. The proliferation rate was detected by cell counting. Representative microscopic images (magnification, ×10) and relative cell counts. (D) MTT assay after application of TTFields for 48 h. (E) 3D colony culture after application of TTFields for 48 h. (F) Sensitivity of liver cancer cells treated with TTFields (1.0 V/cm; 150 kHz) was measured using a colony formation assay. Representative microscopic images (magnification, ×40), *P<0.05, **P<0.01, ***P<0.001 vs. respective controls. TTFields/TTF, tumor-treating fields.

Figure 2.

Effect of TTFields on the apoptosis of liver cancer cells. (A) Analysis of cell death in liver cancer cell lines 72 h after treatment with TTFields (1.0 V/cm; 150 kHz) using a cell death detection kit. **P<0.01, ***P<0.001 vs. respective controls. (B) Analysis of caspase activity in liver cancer cell lines 72 h after treatment with TTFields (1.0 V/cm; 150 kHz) using ELISA. ***P<0.001 vs. respective controls. (C) Cell lysates (30 µg) were immunoblotted with the indicated antibodies. *P<0.05, ***P<0.001 vs. respective controls. (D) Analysis of ROS generation in liver cancer cell lines 72 h after treatment with TTFields (1.0 V/cm; 150 kHz) using an ROS detection kit. **P<0.01, ***P<0.001 vs. respective controls. TTFields/TTF, tumor-treating fields; ROS, reactive oxygen species; PARP, poly (ADP-ribose) polymerase 1.

Figure 3.

TTFields inhibit the migration and invasion of liver cancer cells. (A and B) Tumor cell migration and invasion after 24 h TTFields (1.0 V/cm; 150 kHz) treatment examined using Transwell chamber assays. The number of invading tumor cells that penetrated through the Matrigel/gelatin was counted in five high-intensity fields. Representative microscopic images (magnification, ×40), **P<0.01, ***P<0.001 vs. respective controls. (C) Cell lysates (30 µg) were immunoblotted with the indicated antibodies. TTFields/TTF, tumor-treating fields. *P<0.05, **P<0.01 vs. respective controls.

Figure 4.

TTFields sensitizes liver cancer cells to sorafenib. (A and B) Liver cancer cells were treated with TTFields (1.0 V/cm; 150 kHz), sorafenib (5 µM) or both concurrently for 24 h, and cell viability was determined using a trypan blue exclusion assay. (A) Representative microscopic images (magnification, ×10) (left) and cell counts (right). **P<0.01, ***P<0.001 vs. respective controls. (B) MTT assay. *P<0.05, ***P<0.001 vs. respective controls. (C) Colony forming assay. **P<0.01, ***P<0.001 vs. respective controls. TTFields/TTF, tumor-treating fields.

Figure 5.

Effect of TTFields combined sorafenib on the apoptosis of liver cancer cells. (A) Analysis of cell death in two liver cancer cell lines 72 h after concurrent treatment with TTFields (1.0 V/cm; 150 kHz) and sorafenib (5 µM) using a cell death detection kit. *P<0.05, ***P<0.001 vs. respective controls. (B) Analysis of caspase activity in the two liver cancer cell lines 72 h after treatment with TTFields and sorafenib using ELISA. ***P<0.001 vs. respective controls. (C) Analysis of ROS generation in two liver cancer cell lines 6 h after treatment with TTFields (1.0 V/cm; 150 kHz) using flow cytometry. *P<0.05, **P<0.01 vs. respective controls. TTFields/TTF, tumor-treating fields; NAC, N-acetylcysteine; ROS, reactive oxygen species.