The CCC cell lines HuCCT-1 (19) and TFK-1 (20) represent intrahepatic and extrahepatic cell origin and different grades of advancement [Riken BRC Cell Bank (Tsukuba, Ibaraki, Japan); German Collection of Microorganisms and Cell cultures (DSMZ; Braunschweig, Germany)], respectively. HuCCT-1 and TFK-1 cells were cultured in RPMI medium which was supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml)/streptomycin (100 µg/ml) (both from Biochrom AG, Berlin, Germany), and stable L-glutamine. Cells were incubated at 37°C in a 5% CO₂ humidified atmosphere and harvested once a week at full confluence. L-SFN (Sigma-Aldrich, St. Louis, MO, USA) was diluted in dimethyl sulfoxide (DMSO) to a concentration of 100 mM and stored in aliquots. We used cisplatin (CisPt) (Teva GmbH, Radebeul, Germany) at a concentration of 1 mg/ml. Adequate dilutions were made prior to treatment.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Carl Roth, Karlsruhe, Germany) assay was used to test the viability of the CCC cells. Approximately 3×10³ cancer cells were seeded in each well into 96-well plates. Cells were treated after 24 h pre-incubation with CisPt (5 µM), SFN (10 µM) and their combination (CisPt 5 µM+SFN 10 µM). At the end of each treatment, 10 µl of 1 mg/ml MTT was added to each well and incubated for 4 h at 37°C. After a 4-h incubation period, the medium was aspirated and plates were left to dry for 30 min. The precipitated formazan crystals were dissolved using 2-propanol (VWR International, Darmstadt, Germany) and absorbance at 570 nm was measured using a spectrophotometer (Anthos Mikrosysteme GmbH, Krefeld, Germany).

To calculate the combination index (CI), we used the following equation: CI = AB/A × B, where AB is the percentage of viable cells in the group receiving combination treatment using drugs A and B for different cell lines, and A and B represent the percentage of viable cells for the treatment groups using drug A or B alone, respectively. A CI value >1 indicates an antagonistic effect, a CI value equivalent to 1 indicates an additive effect, a CI value <1 indicates a synergistic effect and a CI value <0.7 is indicative of significant synergy (21).

Cells (2×10⁵) were seeded into 12-well plates and left to adhere overnight. On the next day, medium was replaced with treatment solution for the following 36 h. Apoptotic cells were detected using Annexin V-FITC apoptosis detection kit (BioVision, Mountain View, CA, USA). Briefly, cells were harvested and centrifuged at 1,800 rpm for 5 min before being washed twice with binding buffer. Then, the cells were incubated in binding buffer containing Annexin V and propidium iodide. For flow cytometric analysis, we used FACSCalibur (Becton-Dickinson Biosciences, Franklin Lakes, NJ, USA). At least 10,000 cells were gated for each experiment. The total numbers of cells in the right upper and lower quadrants were counted as apoptotic cells.

To assess how much DNA was platinated, we seeded 2×10⁵ cells into 12-well plates. After 24 h, we added the treatment solution and incubated for an additional 48 h. Cells were then harvested, centrifuged, and fixed with 4% paraformaldehyde on ice, followed by permeabilization with 0.1% Triton X-100 on ice. After permeabilization, primary anti-cisplatin modified DNA (ab103261; ratio 1:200; Abcam, Cambridge, MA, USA) antibody was added before being dissolved in phosphate-buffered saline (PBS) containing 1% FCS. Cells were then washed twice after a 1-h incubation and secondary Alexa Flour 488-conjugated anti-rat IgG (Cell Signaling Technology, Beverly, MA, USA) antibody was added in a ratio of 1:1,000. After 30 min of incubation in the dark, the cells were analyzed using FACSCalibur (Becton-Dickinson).

After 48 h of cell culture incubation with treatment, cell lysates were prepared in RIPA buffer (Sigma-Aldrich) using a proteinase inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). NuPAGE 4–12% Bis-Tris, NuPAGE 12% Bis-Tris and NuPAGE 4% Tris-Gly Gels (Novex, Carlsbad, CA, USA) electrophoresis of 20 µg of each protein sample was performed using XCell SureLock Mini-Cell Module (Invitrogen, Carlsbad, CA, USA). Then, the cells were transferred to nitrocellulose membranes (Bio-Rad Laboratories, Munich, Germany) using XCell II™ Blot Module (Invitrogen). Membranes were blocked in TBS + 0.1% Tween with 5% BSA (Serva Electrophoresis, Heidelberg, Germany) and incubated with primary antibodies at 4°C overnight, followed by incubation with secondary antibodies for 1 h at room temperature. All membranes were scanned using LI-COR Odyssey CLx scanner (LI-COR Biotechnology, Lincoln, NE, USA). All primary were purchased from Cell Signaling Technology, unless otherwise indicated. Primary antibodies were diluted to a ratio of 1:1,000 and secondary antibodies to a ratio of 1:10,000 in 5% BSA solution. Western blot quantification was performed with LI-COR Image Studio Software (LI-COR Biotechnology).

All data are expressed as mean ± SD and were evaluated with one-way ANOVA using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA). Each experiment was repeated at least three times. Values were considered significant at p<0.05.

MTT viability assay was used to assess the effect of SFN, CisPt and their combination therapy on the CCC cell lines, TFK-1 and HuCCT-1 (Table I). Treatment was shown to reduce the viability of TFK-1/HuCCT-1 cells in a time-dependent manner to 26.7±5.53/18.13±3.56, 57.72±8.92/65.25±11.12 and 11.04±3.78/5.11±2.00%, respectively after 72 h. The CI was 0.72 and 0.43 in the TFK-1 and HuCCT-1 cells, respectively (Fig. 1).

To examine the effect on apoptosis, the cells were treated, followed by staining with Annexin V, propidium iodide and FACS analysis 36 h later. While the treatment with either SFN or CisPt alone did not induce significant apoptosis at this early time point, the combination treatment significantly increased the percentage of apoptotic cells from 7 to 16%, and from 13 to 23% in the TFK-1 and HuCCT-1 cell lines, respectively (Fig. 2). Correspondingly, the expression of cleaved caspase-3 and cleaved PARP was greater after the combination therapy compared to the expression levels noted in the controls and single substances 48 h after treatment (Fig. 3).

To determine whether SFN mediated the observed effect by modulating the DNA damage response, we examined the expression of a panel of proteins known to be involved in DNA damage response. In most cases, only CisPt led to a pronounced induction in the phosphorylated forms of ATM, ATR, H2A.X (a genuine marker of DNA damage) (22) Chk1, Chk2 and p53, whereas the combination treatment largely did not increase the protein levels that were already enhanced by the presence of CisPt alone (Fig. 4A). In order to elucidate whether SFN may have increased the percentage of DNA, platinated by CisPt, we detected platinated DNA by staining with a specific antibody and FACS analysis. However, we did not detect a statistically significant increase in the amount of platinated DNA upon the combination of CisPt and SFN (Fig. 4B), thereby suggesting that SFN may sensitize the cells by influencing apoptosis mechanisms.

To further explain the assumption that SFN targets apoptosis signaling, we examined the expression of the anti-apoptotic genes Bcl-2 and XIAP and pro-apoptotic Bax via western blot analysis. CisPt induced the expression of Bcl-2 and this induction was inhibited by the combination with SFN (Fig. 5). A similar tendency was observed for the expression of XIAP. Bax expression was not altered by any of the treatment groups.