Differential effectiveness of tyrosine kinase inhibitors in 2D/3D culture according to cell differentiation, p53 status and mitochondrial respiration in liver cancer cells

Abstract

Sorafenib and Regorafenib are the recommended first- and second-line therapies in patients with advanced hepatocellular carcinoma (HCC). Lenvatinib and Cabozantinib have shown non-inferior antitumoral activities compared with the corresponding recommended therapies. The clinical trials have established recommended doses for each treatment that lead different blood concentrations in patients for Sorafenib (10 µM), Regorafenib (1 µM), Lenvatinib (0.1 µM), and Cabozantinib (1 µM). However, very low response rates are observed in patients attributed to intrinsic resistances or upregulation of survival signaling. The aim of the study was the comparative dose-response analysis of the drugs (0-100 µM) in well-differentiated (HepG2, Hep3B, and Huh7), moderately (SNU423), and poorly (SNU449) differentiated liver cancer cells in 2D/3D cultures. Cells harbors wild-type p53 (HepG2), non-sense p53 mutation (Hep3B), inframe p53 gene deletion (SNU423), and p53 point mutation (Huh7 and SNU449). The administration of regular used in vitro dose (10 µM) in 3D and 2D cultures, as well as the dose-response analysis in 2D cultures showed Sorafenib and Regorafenib were increasingly effective in reducing cell proliferation, and inducing apoptosis in well-differentiated and expressing wild-type p53 in HCC cells. Lenvatinib and Cabozantinib were particularly effective in moderately to poorly differentiated cells with mutated or lacking p53 that have lower basal oxygen consumption rate (OCR), ATP, and maximal respiration capacity than observed in differentiated HCC cells. Sorafenib and Regorafenib downregulated, and Lenvatinib and Cabozantinib upregulated epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor receptor (c-Met) in HepG2 cells. Conclusions: Sorafenib and Regorafenib were especially active in well-differentiated cells, with wild-type p53 and increased mitochondrial respiration. By contrast, Lenvatinib and Cabozantinib appeared more effective in moderately to poorly differentiated cells with mutated p53 and low mitochondrial respiration. The development of strategies that allow us to deliver increased doses in tumors might potentially enhance the effectiveness of the treatments.

Fig. 1. Drug effectiveness in liver cancer cells cultured in spheroids.

Effect of Sorafenib, Regorafenib, Lenvatinib, and Cabozantinib in the area of spheroids generated by HepG2 (a), Hep3B (b), and Huh7 (c) cells. Drugs (10µM) were administered at day 8th after spheroid establishment, and cultures were maintained up to day 15th as described in “Materials and methods” section. The area of the spheroids (µm², %, fold over control) were measured at days 8th, 10th, 12th, and 15th. All results are expressed as mean±SD of independent experiments (n = 3). The groups with statistically significant differences among them (p ≤ 0.05) were indicated with different letters (a, b, c, d, e, or f). Magnification of images are ×10.

Fig. 2. Drug effectiveness in HepG2 cells cultured in spheroids.

Effect of Sorafenib, Regorafenib, Lenvatinib, and Cabozantinib in non-trypan blue-stained viable cells (a), trypan blue non-viable cells (b), Ki67-positive cells (c), caspase-3 activity (d), and TUNEL-positive cells (e) in spheroids generated by HepG2 cells. Drugs (10µM) were administered at day 8th after spheroid establishment, and cultures were maintained up to day 15th as described in “Material and methods” section. The parameters were measured at days 10th and 15th. Trypan blue staining in cells from trypsin-dissociated spheroids allowed the identification of viable and non-viable cells (%, fold over control). Ki67- and TUNEL-positive cells were determined by immunohistochemistry, and caspase-3 activity was assessed using commercial caspase-Glo® Assay Systems as described in “Materials and methods” section. Ki67- and TUNEL-positive cells were assessed by fluorescence methods (fluorescence, %, fold over control). Caspase-3 activity is shown as the RLU (%, fold over control). All results are expressed as mean±SD of independent experiments (n = 3). The groups with statistically significant differences among them (p ≤ 0.05) were indicated with different letters (a, b, c, d, or e).

Fig. 3. Drug effectiveness in liver cancer cells cultured in monolayer.

Effect of Sorafenib, Regorafenib, Lenvatinib, and Cabozantinib in BrdU incorporation (a) and caspase-3 activity (b) in HepG2, Hep3b, and Huh7 cells cultured in 2D system. Drugs (10µM) were administered at 24h after plating. BrdU incorporation and caspase-3 activity were determined 24h after drug administration using commercial colorimetric assay and caspase-Glo® Assay Systems as described in “Materials and methods” section, respectively. BrdU incorporation is shown as the absorbance at 370nm (reference wavelength: 492nm; absorbance, %, fold over control). Caspase-3 activity is shown as the RLU (%, fold over control). All results are expressed as mean ± SD of independent experiments (n = 4). The groups with statistically significant differences among them (p ≤ 0.05) were indicated with different letters (a, b, c, d, e, f, or g).

Fig. 4. Drug effectiveness on cell proliferation in liver cancer cells cultured in monolayer.

Effect of Sorafenib (a), Regorafenib (b), Lenvatinib (c), and Cabozantinib (d) in BrdU incorporation in HepG2, Hep3b, Huh7, SNU423, SNU449, and primary human hepatocytes cultured in 2D system. Graphs are separated according to treatments. Drugs (0, 100nM, 1µM, 10µM, and 100µM) were administered at 24h after plating. BrdU was determined 24h after drug administration using a commercial colorimetric assay, as described in as described in “Materials and methods” section. BrdU incorporation is shown as the absorbance at 370nm (reference wavelength: 492nm; absorbance, %, fold over control). All results are expressed as mean±SD of independent experiments (n = 6). The groups with statistically significant differences among them (p ≤ 0.05) were indicated with different letters (a, b, c, d, or e).

Fig. 5. Drug effectiveness on apoptosis in liver cancer cells cultured in monolayer.

Effect of Sorafenib (a), Regorafenib (b), Lenvatinib (c), and Cabozantinib (d) in caspase-3 activity in HepG2, Hep3b, Huh7, SNU423, SNU449, and primary human hepatocytes cultured in 2D system. Graphs are separated according to treatments. Drugs (0, 100nM, 1µM, 10µM, and 100µM) were administered at 24h after plating. Caspase-3 activity was determined 24h after drug administration using a commercial caspase-Glo® Assay Systems as described in “Materials and methods” section. Caspase-3 activity is shown as the RLU (%, fold over control). All results are expressed as mean±SD of independent experiments (n = 6). The groups with statistically significant differences among them (p ≤ 0.05) were indicated with different letters (a, b, c, d, or e).

Fig. 6. Drug effectiveness on cell proliferation receptor protein expression in HepG2 cells cultured in monolayer.

Effect of Sorafenib, Regorafenib, Lenvatinib, and Cabozantinib in the protein expression of EGFR and c-Met in HepG2 cells cultured in 2D system. Drugs (10µM) were administered at 24h after plating. The expression of tyrosine kinase receptors was assessed 24h after drug administration by SDS–PAGE electrophoresis coupled to western blot procedure as described in “Materials and methods” section. The values were obtained by densitometric analysis of the spots in relation to their loading control in the blots (densitometry, %, fold over control). All results are expressed as mean±SD of independent experiments (n = 3). The level of significance was set at *p ≤ 0.05 and **p ≤ 0.01 in comparison with their corresponding control.