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

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.


Introduction
Vascular endothelial growth factor (VEGF) promotes angiogenesis in hepatocellular carcinoma (HCC). VEGF is upregulated in HCC as compared with surrounding non-HCC tissues (1); this upregulation has been correlated with advanced stage and poor outcome in HCC (2). Sorafenib, a multikinase inhibitor administered orally to HCC patients, targets the VEGF receptor, platelet-derived growth factor receptor and c-kit (3). Sorafenib treatment has been demonstrated to significantly prolong the survival times of HCC patients: 10.7 months as compared with 7.9 months in a placebo group (4).
However, one limitation of sorafenib treatment is the resistance of HCC to the reagent. Phosphatidyl-inositol (PI) 3 kinase and mitogen-activated protein (MAP) kinase are predominant downstream signaling pathways of VEGF that regulate cell proliferation (5). Although sorafenib inhibits the MAP kinase signaling pathway (6), the PI3 kinase signaling pathway is not affected, thereby resulting in HCC resistance (7). Another limitation of sorafenib is toxicity, which negatively affects the patient's quality of life. For example, a high rate of dermatological adverse effects has been reported (4,8). However, administering a combination of sorafenib and other molecular targeting agents is expected to improve the efficacy and relieve particular adverse effects of the drug. For example, liver-specific microRNA-122 sensitizes tumors to the antitumor effects of sorafenib (9). However, a major limitation of microRNA is that the effects depend on transfection efficiency; untransfected cells are not affected. Therefore, small molecule inhibitors are desirable as these inhibitors affect the majority of cells.
Insulin-like growth factor (IGF)-1 is a hormone that is expressed abundantly in the fetus and exerts an important role in fetal growth and development. Inhibiting the IGF-1 signaling pathway in cancer therapy may have no adverse effects, since IGF-1 concentrations are reduced following birth (10). Picropodophyllin (PPP) is a specific inhibitor of the IGF-1 receptor (IGF-1R), which is involved in tumor cell growth (11,12). PPP has been shown to successfully suppress the proliferation of HCC and hepatoblastoma cells (13,14).
Therefore, in the present study, the proliferation and motility of HCC cells that had been treated with a combination of PPP and sorafenib were analyzed. Normal human umbilical vein endothelial cells (HUVECs) were also used to assess angiogenesis following drug treatment.

Materials and methods
Cell culture. HLF and PLC/PRF/5 HCC lines were purchased from the RIKEN cell bank (RIKEN Life Science Center, Tsukuba, Japan) and cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY, USA). HUVECs (Lonza, Basel, Switzerland) were cultured in EGM™-2 BulletKit™ (Lonza) following the manufacturer's instructions. The cultured cells were incubated in 5% carbon dioxide at 37˚C in a humidified chamber. Hematoxylin and eosin (H&E) staining was performed on cells grown in four-well chambers (Becton Dickinson, Franklin Lakes, NJ, USA) after 48 h of incubation.
Scratch assay. The HUVEC cells were injured using a sterile 200-µm pipette tip at 24 h after plating into four-well chambers; the cells were then stained with H&E after 48 h (15). The distance between the scratched line and the growing edge of the cells was measured at five points.
Statistical analysis. One-way analysis of variance was utilized for statistical analysis using JMP10.0.2 (SAS Institute, Cary, NC, USA). P<0.05 was considered to indicate a statistically significant difference.
H&E staining. HLF ( Fig. 2A and B), PLC/PRF/5 ( Fig. 2C and D) and HUVEC (Fig. 2E and F) cells were stained with H&E to assess the morphological changes following drug treatment. The cells that had been treated with 3 µM sorafenib and 0.2 µM PPP exhibited pyknotic nuclei, which is a characteristic of apoptotic cells (Fig. 2B, D and F). Pyknotic nuclei were not observed in the untreated cells ( Fig. 2A, C and E). Scratch assay. The motility of HLF and PLC/PRF/5 cells was analyzed using a scratch assay (Fig. 3A-D). The distance between the scratched line and the growing edge of the cells was measured for cells with (Fig. 3B and D) or without ( Fig. 3A and C) treatment with 3 µM sorafenib and 0.2 µM PPP. Cell motility was significantly suppressed by the treatment, as compared with that of the control (P<0.05; Fig. 3E).

Discussion
In the present study, PPP enhanced sorafenib-induced suppression of proliferation and motility in HCC cells. NVP-AEW541, another IGF-1R inhibitor, and sorafenib were previously shown to suppress cell proliferation and induce apoptosis synergistically (16). These data suggest that IGF-1R inhibitors and sorafenib suppress cell proliferation synergistically. Sorafenib upregulates IGF-1R and increases Akt (Ser473) phosphorylation (17,18). This suggests that sorafenib may activate signaling pathways downstream of IGF-1R; thus, treating HCC cells with IGF-1R inhibitors and sorafenib is feasible.    Data are presented as the mean ± standard deviation cell proliferation as a percentage of the untreated control cell proliferation. a Variables lower than those treated with sorafenib at 10 µM alone, n=3. PPP, picropodophyllin. Table II  Data are presented as the mean ± standard deviation cell proliferation as a percentage of the untreated control cell proliferation. a Variables lower than those with sorafenib at 10 µM alone, n=3. PPP, picropodophyllin.
Suppressing angiogenesis is a predominant mechanism of the antitumor effects of sorafenib (21). Similarly, inhibiting IGF-1R was shown to suppress the proliferation of HUVECs and induce apoptosis (22). The present study clearly demonstrated that the combination of sorafenib and PPP markedly suppressed proliferation and induced apoptosis in HUVECs. This suggests that the combination of PPP and sorafenib may more effectively suppress angiogenesis.
The combination of 1 µM NVP-AEW541 and 10 µM sorafenib has been previously demonstrated to suppress cell proliferation more effectively than 10 µM sorafenib alone (16). However, the combination of NVP-AEW541 and concentrations of sorafenib lower than10 µM has not been investigated. In the present study, the combination of 0.2 µM PPP and 3 µM sorafenib decreased cell proliferation more efficiently than 10 µM sorafenib alone. This suggests that using co-treatment with an IGF-1R inhibitor may allow the effective dose of sorafenib to be reduced, which may lower the risk of adverse effects. Nevertheless, the combination of PPP and sorafenib may cause different adverse events. Thus, future studies that analyze, through western blotting, the signaling pathways that are altered by co-treatment are required.
In conclusion, in the present study, PPP enhanced sorafenib-induced suppression of proliferation and motility in HCC cells. Therefore, the combination of PPP and sorafenib may exert antitumor and antiangiogenic effects.