Cisplatin and ultra-violet-C synergistically down-regulate receptor tyrosine kinases in human colorectal cancer cells

Bromodeoxyuridine (BrdU) is a synthetic thymidine analog that gets incorporated into DNA during cell division. Therefore, the measurement of BrdU incorporation reflects the ability of cell growth. We first investigated the effects of cisplatin and/or UV-C on cell proliferation using BrdU. Whereas either 10 μM of cisplatin or 10 J/m² of UV-C hardly affected BrdU incorporation in SW480 and DLD-1 cells (Figure 1A, lanes 2 and 3, respectively), the combination caused a marked inhibition in BrdU incorporation (Figure 1A, lane 4, respectively). While it has previously been reported that cisplatin induces cell cycle arrest at the G2-phase [27], cell cycle analysis using flow cytometry revealed that the combination of cisplatin and UV-C increased the population at G2/M phases (28.2 ± 1.35%), compared with cisplatin (21.9 ± 0.68%; p = 0.0014) or UV-C (15.2 ± 0.76%; p = 0.0004) (Figure 1B). Moreover, we examined the protein level of phospho-Rb and cyclin D1, both of which direct cells toward proliferation by controlling progression through the restriction point of cell cycle (Figure 2A) [28]. In SW480 cells, cisplatin by itself had little effect on phosphorylation level of Rb. However, when the cells were first exposed to UV-C and then incubated in the presence of cisplatin, the protein level of phospho-Rb was decreased in a time-dependent manner after 12 h (Figure 2). Since we have recently reported that 10 J/m² of UV-C did not cause the decrease in the protein level of Rb [26], these results suggest that the combination of cisplatin and UV-C exerts synergistic effect on the suppression of cell cycle. We also verified the combination effect in DLD-1, HT29 and HCT116, other human colorectal cancer cell lines (Figure 2).

We next performed colony formation assay, which is a microbiology technique for studying the effectiveness of specific agents on the survival and proliferation of cells (Figure 2B) [29]. The combination synergistically suppressed colony formation of SW480 cells, although cisplatin or UV-C alone did to a lesser extent. Similarly, the combination synergistically decreased the number of colony formation in DLD-1 and HCT116 cells, whereas UV-C alone slightly affected them in these cells. As for HT29 cells, while cisplatin or UV-C alone has no effect, the combination synergistically suppressed colony formation. As a whole, these results suggest that the combination has cytocidal effects on several colorectal cancer cells.

We next investigated the combination effect of cisplatin and UV-C on apoptosis by observing PARP cleavage, since PARP is a family of proteins involved in a number of cellular processes involving mainly DNA repair and programmed cell death, indicating cell apoptosis [30]. While cisplatin or UV-C alone had little effect on PARP, the combination caused PARP cleavage in SW480, DLD-1, HT29 and HCT116 cells (Figure 3A). While Hoechst33258 are used to stain DNA and easily detect such DNA fragments, we next examined the effect of combination of cisplatin and UV-C on DNA fragmentation utilizing this dye and found that the combination increased the number of Hoechst 33258-positive apoptotic cells in SW480 and HT29 cells (Figure 3B), which are consistent with our results shown in Figure 3A.

As described in Introduction, EGFR downregulation is the most prominent regulatory system in signal attenuation and involves the internalization and subsequent degradation of the activated receptor in the lysosomes. As well, HER2 is frequently overexpressed in colorectal cancer when compared with normal colonic mucosa, and the extent of overexpression seems to correlate with increasing disease stage and poorer patient survival [31]. Therefore, therapies that target the EGFR and/or HER2 may be effective in the chemoprevention and/or therapy of colorectal cancer [32]. Whereas we recently reported that EGFR signaling plays a critical role in proliferation of colorectal cancer cells [26], we next focused on the expression level of EGFR as well as HER2 in several colorectal cancer cells including SW480, DLD-1, HT29 and HCT116, since we observed that the combination use of cisplatin and UV-C synergistically exerts suppressive effect on cell proliferation and apoptosis (Figures 1 and 3). As depicted in Figure 4, 10 μM cisplatin alone did not affect these levels even after a longer treatment in SW480 (Figure 4, lanes 1–4). As well, while UV-C at a dose over 30 J/m² caused a marked decrease in the EGFR protein level [26], in this study we observed that 10 J/m² of UV-C did not affect (Additional file 1). Interestingly, the combination use of 10 μM cisplatin and 10 J/m² UV-C clearly induced the decrease in the protein levels of EGFR as well as HER2 in SW480 cells, which were appeared at 12 h after treatment with cisplatin and UV-C (Figure 4, lanes 5–8). Similar results were observed in other colorectal cancer cells, DLD-1, HT29 and HCT116. Together, the combination effect of cisplatin and UV-C on the suppression of cell growth seems to be due to the down-regulation of EGFR and/or HER2.

It has previously been reported that UV irradiation (100 J/m²) induces rapid and persistent internalization of EGFR [33]. As well, we have recently reported that UV-C at a dose over 30 J/m² caused the internalization and subsequent down-regulation of the EGFR in SW480 cells [26]. In order to elucidate the mechanism underlying combination effect of cisplatin and UV-C, we next examined whether cisplatin (10 μM) and/or UV-C (10 J/m²) induces changes in the subcellular localization of EGFR in SW480 cells. Whereas antibody-tagged EGFR remained on the cell surface (Figure 5A, panels 1, 6 and 11), 0.5 h incubation after the treatment of the cells with UV-C alone (10 J/m²) resulted in the distribution of the EGFR to cytosol beneath the plasma membrane, thus indicating that UV-C indeed induced the internalization of the EGFR (Figure 5A, panel 7). By contrast, cisplatin (10 μM) by itself did not affect the localization of the EGFR (Figure 5A, panels 2–5). Interestingly, when the cells were first exposed to UV-C and then incubated in the absence of cisplatin for 6 h and more, the antibody-tagged EGFR reappeared on the cell surface, thus suggesting that internalized EGFR recycled back to the cell membrane (Figure 5A, panels 8–10). However, the EGFR remained to be internalized when the cells were treated with the combination of cisplatin and UV-C (Figure 5A, panels 12–15).

To verify these results, we measured the amount of cell surface EGFR by enzyme-linked immunosorbent assay (ELISA). Whereas UV-C alone decreased the amount of cell surface EGFR within 0.5 h (Figure 5B, lane 3). However, they were gradually recovered 3 h after treatment with UV-C (Figure 5B, lanes 3, respectively). On the contrary, cell surface EGFR in the cells treated with the combination of cisplatin and UV-C remained to be decreased (Figure 5B, lanes 4, respectively). Taken together with our results obtained from fluorescence study, we strongly suggest that the treatment with cisplatin after UV-C exposure blocks the recycling of the EGFR which are internalized by UV-C.

SW480 and HT29 human colorectal cancer cells, that were obtained from American Type Culture Collection (Manassas, VA), were grown in Dulbecco's modified Eagle’s medium (DMEM) (Invitrogen, San Diego, CA), containing 10% fetal calf serum (FCS) with penicillin (100 U/ml) and streptomycin (100 μg/ml) in a humidified 5% CO₂ incubator at 37°C. DLD-1 and HCT 116 human colorectal cancer cells were from American Type Culture Collection (Manassas, VA) and grown in Roswell Park Memorial Institute 1640 (RPMI) (Invitrogen, San Diego, CA) as described above.

UV-C exposure of cells was performed in UV-C 500 UV Crosslinker (8 W 254 nm UV lamp) (GE Healthcare), which creates CW light using 8 W 254 nm UV lamps. Fluorescent lamps without a phosphorescent coating emit UV with two peaks at 254 nm and 185 nm due to the peak emission of the mercury within the bulb. UV lamps used quartz (glass) block the 185 nm wavelength and emit only 254 nm UV. After aspiration of the growth medium, the cells were exposed to the indicated dose (J/m² = 100 μJ/cm²) of UV-C in 5 sec, and then incubated for the indicated times.

BrdU incorporation was measured using cell Proliferation ELISA (BrdU). The cells (7 x 10³/well) were seeded onto 96-well plates and 48 h later, the cells were exposed to the indicated doses (0 or 10 J/m²) of UV-C, just after the aspiration of the growth medium. The cells were then incubated in DMEM or RPMI medium with 1% FCS and 10 μM of cisplatin for 24 h. They were then used for the assay according to the manufacturer’s protocol. All assays were done at least three times.

Cell cycle analysis was done as described previously [7]. In brief, SW480 cells were exposed to UV-C, followed by the incubation in DMEM with/without 10 μM of cisplatin for 96 h. The cells were then harvested and stained with 500 μl of PI/RNase staining buffer for 15 min at room temperature. They were finally analyzed by flow cytometry using a FACS Calibur instrument (Becton Dickinson); data were analyzed using the CELL Quest computer program (Becton Dickinson) as previously described. All data were obtained from at least three independent experiments.

Human colorectal cancer cells (SW480, DLD-1, HT29 and HCT116) were exposed to UV-C and then incubated in DMEM or RPMI medium and with/without 10 μM of cisplatin. Twenty four h after treatment, the cells were trypsinized and the cells (3 x 10³) were re-seeded into fresh tissue culture dishes and incubated for 7 days. Fresh media were added at day 4. At day 7, the media were removed and the cells were fixed with 2 ml of clonogenic reagent (50% ethanol, 0.25% 1,9-dimethyl-methylene blue) for 45 min. They were then washed with PBS twice and counted the blue colonies on 5 randomly chosen fields.

The cells were lysed in lysis buffer [20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% TritonX-100, 2.5 mM sodium pyrophosphate, 50 mM NaF, 50 mM HEPES, 1 mM Na₃VO₄ and 2 mM phenylmethylsulfonyl fluoride (PMSF)] and scraped from the Petri dishes. Protein extracts were examined by Western blot analysis as previously described [49, 50].

Immunofluorescence microscopy studies were performed as described previously [46]. Live cells grown on coverslip-bottom dishes in DMEM were first exposed to the mouse anti-EGFR antibody that recognized the extracellular domain of EGFR for 15 min and then exposed to UV-C (10 J/m²) and/or cisplatin (10 μM) and incubated in DMEM for the indicated times (0.5 h, 6 h, 12 h and 24 h) at 37°C. They were then fixed with 4% paraformaldehyde for 10 min on ice and then exposed to 0.1% Triton X-100 for 10 min to permeabilize the cell membrane. They were followed by exposure to Alexa Fluor 488® conjugated donkey anti-mouse IgG antibodies (green signal) and 4',6-diamidino-2-phenylindole (DAPI) for 1 h. The cells were then examined by fluorescence microscopy, BIOREVO (BZ-9000) (Keyence, Tokyo, Japan) according to the manufacturer’s protocol.

Quantification of cell surface EGFR was performed as described previously [26]. In brief, SW480 cells were first exposed to the mouse anti-EGFR antibody in DMEM containing 1% BSA, for 15 min at 37°C. The cells were then incubated for the indicated times in DMEM with/without 10 μM of cisplatin after exposure to UV-C, then fixed with 4% paraformaldehyde for 10 min on ice. After blocking with 1% BSA in PBS for 1 h, the cells were exposed to an anti-mouse IgG, horseradish peroxidase-linked whole antibody (GE healthcare, Piscataway, NJ) for 1 h at room temperature, followed by washing four times with PBS containing 1% BSA. Finally, the cells were exposed to 50 μl of 1-stepTM Ultra TMB-ELISA reagent (Pierce, Rockford, IL) for 5 min at room temperature. The absorbance of each sample at 450 nm was then measured.

Live cells grown on coverslip-bottom dishes were first exposed to UV-C (10 J/m²) and/or cisplatin (10 μM) for 72 h and then stained with Hoechst 33258 in DMEM without FCS for 1 h at 37°C. They were then fixed with 4% paraformaldehyde for 10 min on ice. The cells were then examined by fluorescence microscopy, as described above.

The densitometric analysis was performed using scanner and image analysis software (Image J ver. 1.45 g). The back ground subtracted signal intensity of each protein signal was normalized by the respective control signal. All data were obtained from at least three independent experiments.

The data were analyzed by ANOVA followed by Bonferroni method for multiple comparisons between the indicated pairs, and a p < 0.05 was considered significant.