Picropodophyllin and sorafenib synergistically suppress the proliferation and motility of hepatocellular carcinoma cells

Abstract

Resistance is one limitation of sorafenib in the treatment of hepatocellular carcinoma (HCC). Insulin-like growth factor-1 receptor (IGF-1R) is involved in cancer cell proliferation. To assess the potential synergistic antitumor effects of picropodophyllin (PPP), an IGF-1R inhibitor, HLF and PLC/PRL/5, HCC cells were treated with PPP alone or PPP in combination with sorafenib, a multikinase inhibitor. Normal human umbilical vein endothelial cells (HUVECs) were also used to analyze the antiangiogenic effects of the drugs. HCC cells and HUVECs were cultured on 96-well plates, and then treated with PPP, with and without the addition of sorafenib. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay and hematoxylin and eosin staining were then performed 48 h later. The HCC cells were also analyzed using scratch assays and hematoxylin and eosin staining after 48 h. The proliferation of HLF, PLC/PRF/5 and HUVEC cells was suppressed by the combination of 0.2 μM PPP and 3 μM sorafenib more effectively than by 10 μM sorafenib alone. The motility of HLF and PLC/PRF/5 cells was also suppressed to a greater extent with the combination of PPP at 0.2 μM and sorafenib at 3 μM than with sorafenib at 10 μM alone. The cells that had been treated with 0.2 μM PPP and 3 μM sorafenib also exhibited pyknotic nuclei, which is characteristic of apoptosis. In conclusion, PPP enhanced sorafenib-mediated suppression of proliferation and motility in HCC cells. Therefore, the combination of PPP and sorafenib may exert antitumor and antiangiogenic effects.

Keywords: insulin-like growth factor 1 receptor; scratch assay.

Figure 1

Cell proliferation assay. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay was performed following the addition of picropodophyllin (PPP) and/or sorafenib (Sora) to (A) HLF, (B) PLC/PRL/F and (C) normal human umbilical vein endothelial cells, and the cell proliferation rate is presented as the percentage of the untreated cell proliferation rate. ●, 0 μM PPP; ■, 0.02 μM PPP; ○, 0.06 μM PPP; □, 0.2 μM PPP; ×, 0.6 μM PPP; (−), without PPP; (+), with PPP, n=3.

Figure 2

Hematoxylin and eosin staining. (A and B) HLF, (C and D) PLC/PRF/5 and (E and F) normal human umbilical vein endothelial cells were cultured on chamber slides. The cells were stained using hematoxylin and eosin following two days incubation with (B, D and F) or without (A, C and E) the addition of picropodophyllin (0.2 μM) and sorafenib (3 μM). Arrow, apoptotic cells with pyknotic nuclei; original magnification, ×400; scale bar, 50 μm.

Figure 3

Scratch assay. (A and B) HLF or (C and D) PLC/PRL/5 cells were cultured on chamber slides. The cells were scratched using a 200-μl pipette tip (solid line), and treated with (B and D) or without (A and C) picropodophyllin (0.2 μM) and sorafenib (3 μM). (E) The distance between the scratched line and the growing edge of the cells was measured. Original magnification, ×100; scale bar, 200 μm; error bars indicate standard deviations. (−), cells cultured without picropodophyllin or sorafenib; (+), cells cultured with picropodophyllin and sorafenib. ^*P<0.05, average distance of cells cultured with picropodophyllin and sorafenib compared with cells without picropodophyllin or sorafenib (n=3).