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Introduction

Cholangiocellular carcinoma (CCC) is the second most frequent malignant primary liver tumor accounting for ~15% of all reported cases (1). CCC is classified into intrahepatic, perihilar and extrahepatic carcinoma, accounting for 6–8%, 50–67% and 27–42% of cases, respectively (2). The 5-year survival of newly diagnosed CCC was only 18–19% in 2010 (3). CCC is usually diagnosed at advanced stages and only a small portion of patients are eligible for curative surgery. Even after R0 resection, the 5-year survival is still very low, ranging from 0 to 40% of cases (4). For the majority of patients, chemotherapy with gemcitabine, cisplatin or 5-fluorouracil remains the only therapeutic option. However, even then the average survival is ~6 months (5).

Cisplatin is one of the most effective chemotherapeutic drugs that has been used in cancer treatment for over 30 years. Cisplatin and other platinum-based drugs have been used in monotherapy or in combination for treating various types of cancers, including ovarian carcinomas, lung cancer, various lymphomas, sarcomas and CCC (4,6–9). The main mechanism of action of cisplatin is based on forming DNA adducts and triggering double-strand breaks (10). Although, cisplatin is a very potent anticancer drug, its effectiveness is diminished by the development of increasing resistance to cisplatin (11). Thus, many researchers have focused on identifying strategies to increase the sensitivity of chemotherapeutic agents.

The bioactive agent, sulforaphane (SFN), is an isothiocyanate cleavage product of glucoraphanin that has been transformed by the plant enzyme myrosinase. It is obtained from damaged cruciferous vegetables such as broccoli, cauliflower, cabbage and Brussels sprouts (12,13). In the past 20 years, SFN was proven to be a potent anti-inflammatory, anti-carcinogenic, and chemopreventative agent in many cancers (14). Recent findings suggest that SFN is able to modulate the response to various chemotherapeutic drugs either by increasing sensitivity or reducing resistance of cancer cells to them (15–18). In the present study, we demonstrated for the first time, that SFN not only reduced the viability of human CCC cell lines, but also reduces the resistance to cisplatin and synergistically increases apoptosis, a process which was reflected by the modulation of the expression of proteins involved in apoptosis.

Materials and methods

Cell culture and treatment

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.

Cell viability assay

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).

Drug combination index

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).

Flow cytometric analysis of apoptosis

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.

Platinated DNA content analysis

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).

Western blot analysis

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).

Statistical analysis

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.

Results

Impact of SFN and CisPt on cell viability

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).

Figure 1. Combination of cisplatin and sulforaphane reduces CCC cell viability. The viability of TFK-1 and HuCCT-1 cells was detected using MTT assay 24, 48 and 72 h after sulforaphane (SFN), cisplatin (CisPt) and combination treatment with SFN+CisPt as described in Materials and methods. Data are shown as mean ± SD; ***p<0.001 between groups.

Table I. Drug combination layout indicating the combination indices (CI).

Table I.

Drug combination layout indicating the combination indices (CI).

	CisPt (µM)
CCC cell line	2.5	5
TFK-1 cells
SFN (µM)
5	1.188	0.767
10	0.94	0.722
HuCCT-1 cells
SFN (µM)
5	1.692	1.284
10	0.834	0.4355

[i] Effect of combined treatment on CCC cell lines. MTT assay was used to calculate combination index (CI). CI >1, antagonistic effect; CI <1, additive effect; CI <0,7, synergistic effect. SFN, sulforaphane; CisPt, cisplatin; CCC, cholangiocarcinoma.

Apoptosis

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).

Figure 2. Effect of sulforaphane on apoptosis. (A) Annexin V-FITC was used to quantify the percentage of apoptotic cells 36 h after treatment with sulforaphane (SFN), cisplatin (CisPt) and SFN+CisPt. (B) The total percentages of apoptotic (early and late apoptotic) cells were calculated and are shown as mean ± SD; *p<0.05 between the treatment and control group.

Figure 3. Effect of sulforaphane on the expression of cleaved caspase-3 and cleaved PARP. TFK-1 and HuCCT-1 cells were treated with sulforaphane (SFN), cisplatin (CisPt) and SFN+CisPt, and the expression of cleaved caspase-3 (Asp175, 5A1E) and cleaved PARP (Asp214) were assessed using western blot analysis. β-actin expression was used as a loading control.

DNA damage quantification

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.

Figure 4. Impact of sulforaphane on DNA damage and checkpoint activation. (A) Cell lines were treated with sulforaphane (SFN), cisplatin (CisPt) and SFN+CisPt as described in Materials and methods. The expression levels of phospho(p)-ATM, p-ATR, p-H2A.X, p-P53, p-Chk1 and p-Chk2 were measured using western blotting. (B) Flow cytometry was used to quantify platinated DNA after treatment with CisPt and SFN+CisPt. Data are shown as mean ± SD.

Downregulation of anti-apoptotic proteins can overcome induced resistance

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.

Figure 5. Sulforaphane downregulates anti-apoptotic protein expression in CCC cells. Western blotting was used to compare the expression of anti-apoptotic proteins, Bcl-2 and XIAP, after treatment with sulforaphane (SFN), cisplatin (CisPt) and SFN+CisPt. β-actin expression was used as a loading control.

Discussion

Sulforaphane has been studied for its antioxidant, anti-carcinogenic, chemopreventative and chemosensitizing abilities for over 20 years. It has been shown that sulforaphane enhances the effect of various chemotherapeutic agents in a variety of cancers (16–18,24–26). Data presented in the present study indicate, for the first time, that sulforaphane itself induced apoptosis in two human CCC cell lines which represent intrahepatic and extrahepatic origins of CCC, and enhanced cisplatin-mediated cytotoxicity. However, more detailed analysis in drug synergism is required in future studies.

ATM, ATR and H2A.X play important roles in DNA damage recognition and activation. ATM and ATR are automatically phosphorylated upon DNA damage and this event is followed by phosphorylation of H2A.X (27,28). Phosphorylated H2A.X is considered the hallmark of DNA damage (22,29). The data of the present study indicated that cisplatin increased the expression of the DNA damage-associated proteins, H2A.X, ATM and ATR. Activation of Chk and P53 is the next step in the DNA damage recognition cascade, which is usually followed by cell apoptosis when DNA damage is too extensive (30,31). Indeed, we observed that activation of Chk1 was increased after the combination treatment compared to cisplatin only therapy, but the overall expression of pChk1 was higher in the HuCCT-1 cells. Notably, pChk2 was only upregulated in the TFK-1 cells. From these results, we can speculate that there may be a balance in checkpoint kinase activation. This means, when one kinase is downregulated, the other is upregulated; however, further research is needed to confirm this assumption. We also found that P53 phosphorylation was only present in the HuCCT-1 cells. These findings suggest that the combined treatment of sulforaphane and cisplatin caused extensive DNA damage which may be followed by increased P53 checkpoint activation and apoptosis, thereby causing DNA damage (32).

Several studies indicate that DNA damage correlates with the amount of cisplatin DNA adducts (11,23). To investigate whether increased DNA damage is due to increased DNA adducts, we performed flow cytometry on platinated DNA. The quantities of platinated DNA detected after cisplatin only or the combination therapy were comparable. Thus, other mechanisms may be responsible for the increased DNA damage and apoptosis found after the combination treatment. It has been reported that one of the many mechanisms of resistance to cisplatin involves the overexpression of anti-apoptotic proteins and the Bax/Bcl-2 ratio (11,33). To investigate this hypothesis, we examined the anti-apoptotic proteins Bcl-2 and XIAP by western blot analysis. Cisplatin was found to induce the expression of Bcl-2 in both cell lines, although the expression was higher in TFK-1 cells. However, Bcl-2 expression was downregulated after sulforaphane only or combination therapy. There was no difference in XIAP expression in the TFK-1 cells, whereas it was downregulated in the HuCCT-1 cells after sulforaphane only or in the combination therapy. Expression of pro-apoptotic protein Bax was not altered, which leads to believe that only anti-apoptotic proteins influenced the resistance and the Bax/Bcl-2 ratio. These results suggest that one of the contributing factors in the increased apoptosis and DNA damage was the downregulation of anti-apoptotic proteins, thus enabling the activity of the apoptosis signaling cascade to proceed without interruption (Fig. 6).

Figure 6. Working hypothesis. Proposed signaling pathway of cisplatin combined with sulforaphane.

In conclusion, data shown in the present study clearly indicate, for the first time, that sulforaphane decreased the drug resistance of human CCC cells to cisplatin in vitro via various mechanisms, including a decrease in the levels of anti-apoptotic proteins and increased DNA damage. If the effect of sulforaphane is confirmed in humans, chemotherapy against CCC could be more effective by dietary supplementation and may ultimately increase patient survival.

Acknowledgements

We would like to thank E. Mohr for laboratory and technical advice, and Dr M. Zoeller for technical support. The present study was supported by a research grant of the Heidelberg Surgical Foundation.

References