Exogenous albumin inhibits sorafenib-induced cytotoxicity in human cancer cell lines

Abstract

Sorafenib is an orally administered multikinase inhibitor that exhibits anti-angiogenic and anti-tumor activity. Sorafenib is also known to bind to protein (>99.5%), suggesting protein binding may be involved in sorafenib pharmacokinetic variability. Albumin is a major drug-binding protein. In this study, we examined the effect of albumin on sorafenib-induced cytotoxicity using two in vitro culture cell lines, human hepatoma Huh-7 cells and androgen-independent prostate cancer PC-3 cells. The cells were cultured and incubated, and cytotoxicity was assessed. Results were confirmed by western blotting. The presence of exogenous albumin markedly blocked the sorafenib-induced cytotoxicity in the two cell lines. Albumin concentration, the change of pharmacological signal transduction as Raf-B, vascular endothelial growth factor (VEGF), and phosphorylation of MEK1/2 or ERK1/2 were found to be decreased by sorafenib. Co-incubation of warfarin, a representative coumarin anticoagulant and potent binding activity, with albumin enhanced the cytotoxic effects by sorafenib. These mechanisms depend on the high binding proper ties of sorafenib and exogenous albumin. Furthermore, we investigated the effects of endo genous albumin expression on sorafenib-induced cytotoxicity using the siRNA knockdown system or transfected expression vector assay. However, the cytotoxic effects by sorafenib showed little change either with the knockdown or overexpression of albumin. Our results suggest that particular care should be taken with albuminemia or the combined use of drugs with a high affinity for albumin, such as warfarin, and sorafenib in the treatment of cancer patients. Our findings may be useful to the cancer therapeutic strategy by sorafenib.

Keywords: albumin; cytotoxicity; hepatoma; prostate cancer; sorafenib; warfarin.

Figure 1

Comparison of albumin-supplemented and serum-free media on sorafenib-induced cytotoxicity in (A) human hepatoma Huh-7 cells, or (B) on human prostate cancer PC-3 cells. Each cell line was seeded in a 96-well culture plate overnight under normal culture conditions. The cells were washed in serum-free medium prior to incubation with sorafenib and used for serum-free conditions in this study. Albumin-supplemented conditions were prepared by the addition of albumin (final concentration of albumin at 4%) in serum-free media. The medium was replaced with serum-free conditions and incubated with sorafenib for 48 h. Cytotoxicity was assessed using the MTT assay and survival (%) was calculated relative to the control (sorafenib vehicle) in each condition. Results are the means ± SEM of three individual studies. ^*p<0.05 or ^#p<0.05 compared with the control group, or albumin-supplemented culture condition with each indicated sorafenib incubation, respectively.

Figure 2

Concentration-dependent effect of exogenous albumin on 10 μM sorafenib-induced cytotoxicity in Huh-7 or PC-3 cells. The albumin concentration was changed from 0.1 to 5% in each cell and incubated with sorafenib for 48 h. Cytotoxicity was assessed using the MTT assay and survival (%) was calculated relative to the control (sorafenib vehicle) in each condition. Results are the means ± SEM of three individual studies. ^*p<0.05 compared with the control group (sorafenib vehicle) on indicated albumin-supplemented culture condition.

Figure 3

Western blot analysis of sorafenib-induced changes in the level of pharmacological signal proteins. Cells were incubated with 10 μM sorafenib for 4 h on each indicated concentration of albumin-supplemented condition in Huh-7 or PC-3 cells. Expression of indicated proteins was then analyzed by western blotting using the expression of vinculin, MEK1/2 and ERK1/2 as the controls. Experiments shown are representative of a minimum of three separate experiments.

Figure 4

Effects of warfarin, a representative coumarin anticoagulant on sorafenib-induced cytotoxicity on Huh-7 cells. Warfarin at 0.05-1 mM was co-incubated with 3 μM sorafenib and cultured for 48 h in Huh-7 cells on albumin-supplemented conditions. Cytotoxicity was assessed using the MTT assay and survival (%) was calculated relative to the control (sorafenib vehicle) in each condition. Results are the means ± SEM of three individual studies. ^*p<0.05 or ^#p<0.05 compared to the control group, or each indicated concentration of single incubation with warfarin group, respectively.

Figure 5

Effects of tissue (endogenous) albumin on sorafenib-induced cytotoxicity using (A) a siRNA knock-down system in Huh-7 cells or (B) a transfected expression vector assay in PC-3 cells. Each transfection assay was described in the Materials and methods. Expression of albumin protein was analyzed by western blotting using the expression of β-actin as the control. Experiments shown are representative of a minimum of three separate experiments. Results are the means ± SEM of three individual studies. *p<0.05 or ^#p<0.05 compared to the control and mock groups, respectively.