Phenylbutyrate interferes with the Fanconi anemia and BRCA pathway and sensitizes head and neck cancer cells to cisplatin

To investigate whether phenylbutyrate (PB) sensitizes head and neck cancer cells to cisplatin, three relative cisplatin-resistant head and neck cancer cell lines all expressing wild-type p53 (UM-SCC-1, -6, -25) were used. Cells were treated with 2 mM phenylbutyrate for 5 days, 5 μM cisplatin for 3 days or the combination of the two with cisplatin added on day three. The doses of phenylbutyrate and cisplatin used were chosen because they are clinically achievable. The effects these treatments had on the three cell lines were first analyzed by measuring viability/proliferation using the WST-1 assay. This assay is based on the reduction of the WST-1 reagent by viable cells to produces a soluble formazan salt that can be quantitated with an ELISA plate reader. Since the absorbance correlates with the number of viable cells in the sample, the readout is affected by cell death and inhibition of proliferation during the treatment period. When the three head and neck cancer cell lines were treated with either phenylbutyrate or cisplatin as single agents, viability/proliferation was reduced by 0–10% and 20–30%, respectively (Fig. 1A). When the two treatments were combined, the viability/proliferation was reduced by about 50%, which is more then an additive effect of each agent alone.

Since the WST-1 assay does not distinguish between loss of viability and inhibition of proliferation, we next investigated whether phenylbutyrate could sensitize the cells to cisplatin-induced apoptosis. Cells were treated as for the WST-1 assay described above, collected, fixed and stained with propidium iodide (PI) for the determination of apoptosis using flow cytometry. Treatment with phenylbutyrate alone only marginally increased the percentage of apoptotic cells over mock-treated cells while cisplatin treatment increased apoptosis by 20–30% over control cells (Fig. 1B). When cells were pretreated with phenylbutyrate for 48 hours before adding cisplatin, apoptosis was increased by 40–55% over mock-treated cells suggesting that the effect was more than additive.

Finally, we assessed the potential phenylbutyrate-induced sensitization of the cells to cisplatin using the clonogenic assay. This assay was only applicable to the UM-SCC-1 cell line since the other two cell lines failed to form colonies under our culture conditions. We found that phenylbutyrate did not significantly reduce the clonogenic survival of the UM-SCC-1 cells while cisplatin treatment reduced clonogenic survival by about 20%. When the two treatments were combined, we observed a much more dramatic cell kill than adding up the cell killing effects of the two drugs alone. Taken together, all three cell survival assays used suggest that phenylbutyrate sensitizes head and neck cancer cell lines to cisplatin.

What may be the mechanism by which phenylbutyrate sensitizes these head and neck cancer cell lines to cisplatin? Since cisplatin induces interstrand DNA cross-links and cells deficient in the FA/BRCA pathway are hypersensitive to cisplatin we next explored whether the cisplatin-sensitizing effect of phenylbutyrate correlated to a phenylbutyrate-mediated abrogation of the FA/BRCA pathway. The FA/BRCA response pathway is an important pathway that is thought to orchestrate the processing of DNA interstrand cross-links [4, 5]. A functional readout of an activated FA/BRCA pathway is the formation of microscopic aggregates of FANCD2 proteins in nuclei, most likely representing sites of DNA damage. To investigate whether phenylbutyrate may interfere with the FA/BRCA response pathway and block the formation of FANCD2 nuclear foci after cisplatin treatment, the three different head and neck cancer cell lines were treated with phenylbutyrate, cisplatin or the combination of the two agents. Cells were then fixed and stained with anti-FANCD2 specific antibodies. Fluorescence microscopic inspection of the stained cells showed that cisplatin induced FANCD2 nuclear foci in about 45% of the cells analyzed while phenylbutyrate did not induce any FANCD2 foci over mock-treated control cells (Fig. 2). When the two agents were combined it was clear that phenylbutyrate significantly reduced the number of cells containing cisplatin-induced FANCD2 nuclear foci. Since phenylbutyrate did not affect viability or proliferation of the different cell lines (see Fig. 1A), we do not think the lowering of the number of cells with cisplatin-induced FANCD2 foci by phenylbutyrate is due to a redistribution of the cells in the cell cycle due to the activation of a cell cycle arrest. These results suggest that phenylbutyrate sensitizes head and neck cancer cells to cisplatin by abrogating the FA/BRCA pathway.

Because FANCD2 monoubiquitylation is required for the formation of FANCD2 foci, we next investigated whether phenylbutyrate may affect the cisplatin-induced monoubiquitylation of FANCD2. The three head and neck cancer cell lines were treated with phenylbutyrate, cisplatin or the combination of the two and the induction of monoubiquitylation of FANCD2 was evaluated using Western blot. It was found that cisplatin induced monoubiquitylation of FANCD2 regardless of whether the cells had been pretreated with phenylbutyrate or not (Fig 3). Thus, the phenylbutyrate-mediated abrogation of cisplatin-induced FANCD2 foci formation was not due to a loss of FANCD2 monoubiquitylation.

It has been shown that in addition to monoubiquitylation the formation of FANCD2 nuclear foci following cisplatin treatment requires the tumor suppressor protein BRCA1 [22, 23]. To test whether phenylbutyrate may affect the expression of BRCA1 in head and neck cancer cells, we analyzed the BRCA1 protein levels in mock-treated and phenylbutyrate-treated cells using Western blot. As can be seen in Figure 4A, phenylbutyrate reduced expression of BRCA1 by 48%, 24% and 50% in UM-SCC-1, 6 and 25, respectively. These results suggest that phenylbutyrate may sensitize head and neck cancer cells to cisplatin by interfering with the FA/BRCA pathway through the reduction of expression of BRCA1

If the cisplatin-sensitizing activity of phenylbutyrate is chiefly due to the inhibition of the FA/BRCA1 pathway, then phenylbutyrate should have only limited ability to sensitize head and neck cancer cell lines that are defective in the FA/BRCA1 pathway. To test this possibility, we pre-treated BRCA1-deficient UM-SSC-17B cells [6] with phenylbutyrate and then treated the cells with cisplatin. Since these cells are known to be sensitive to cisplatin we used a lower dose of 2 μM. Although the UM-SSC-17B cells were fairly sensitive to phenylbutyrate, no sensitization to cisplatin was apparent suggesting that phenylbutyrate may only sensitize cells with a functional FA/BRCA1 pathway (Fig. 5, top panels). However, when these experiments were performed using another head and neck cancer cell line (UM-SSC-14A) that also has a defect in the FA/BRCA1 pathway [6], a clear sensitization was observed (Fig. 5, middle panels). The percentage of apoptosis were 12% in controls, 14% for phenylbutyrate alone and 21% for cisplatin alone. When phenylbutyrate and cisplatin treatments were combined the amount of apoptosis rose to 65% which is clearly a more than additive effect. When the same treatment protocol was used on the FA/BRCA proficient cell line UM-SSC-6, we observed an additive effect of the two treatments. Since we used a lower dose of cisplatin in these experiments, the FA/BRCA-proficient cells are not as effected by cisplatin as shown in figure 1 where 5 μM cisplatin was used. It can be seen that both of the FA/BRCA1-defective cell lines show accumulation of cells in late S-phase and G2/M following cisplatin treatment which is in contrast to the similarly treated FA/BRCA1-proficient cell line UM-SSC-6. This is to be expected since the FA/BRCA pathway plays an important role in the coordination of processing of replication-blocking lesions. Taken together, the results suggest that phenylbutyrate do not inhibit the effectivness of cisplatin toxicity in cell lines with a defective FA/BRCA pathway which may have important clinical implications. Furthermore, phenylbutyrate may, in addition to inhibiting the FA/BRCA1 pathway, act on additional targets to sensitize head and neck cancer cells to cisplatin.

The head and neck cancer cell lines UM-SCC-1, -6, -25 were made available to us from the University of Michigan Head and Neck spore program. These cell lines were established from various anatomical locations of head and neck patients. Cisplatin-sensitivity based on the MTT assay has been previously assessed in these cell lines and ID₅₀ for these cell lines were found to be 14.0, 36.7, 18.7 μM respectively [24]. The cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat inactivated fetal bovine serum, penicillin/streptomycin in a humidified 5% CO₂ incubator at 37°C. Cells were treated with 2 mM sodium phenylbutyrate (Scandinavian formulas, PA) for 48 hours and with 5 μM cisplatin (CDDP) (Sigma Chemicals, MO) and incubated for 72 hours.

Exponentially growing UM-SCC-1, -6, -25 cell lines were plated in 96 well plates at a density of 10,000 cells per well and incubated in DMEM at 37°C overnight. Cells were then treated with 2 mM phenylbutyrate for 5 days, 5 μM cisplatin for 3 days or the combination of the two. At the completion of the 5 day incubation, 10 μl of cell proliferation reagent WST-1 (Roche, IN) was added into media in each well and the cells were incubated for 2 hr at 37°C. The absorbance (OD) of each well was determined with a spectrophotometer reading at a wavelength of 490 nm. Absorbance (OD) is assumed to be directly proportional to the number of viable cells.

Determination of the percentage of apoptosis induced following cisplatin treatment was performed as previously described [14, 32]. Cells were treated with 2 mM phenylbutyrate for 5 days, 5 μM cisplatin for 3 days or the combination of the two. After incubation at 37°C, both floating and attached cells (trypsinized) were collected by centrifugation (1500 rpm for 5 minutes) and rinsed with PBS twice. To fix the cells, 500 μl of ice-cold 70% ethanol was added under mixing. After fixing cells for 30 minutes, cells were collected by centrifugation at 1500 rpm and rinsed with PBS twice. Cell pellets were resuspended in 500 μl of propidium iodide (PI) and incubated for 30 minutes at 4°C to stain cellular DNA. Cells with sub-G₁ content of DNA were scored as apoptotic using flow cytometry (Coulter Elite ESP Cell sorter, FL) and the Multicycle software package (Phoenix Flow Systems, CA).

Cells were treated with 2 mM phenylbutyrate for 5 days, 5 μM cisplatin for 3 days or the combination of the two. At the completion of the 5 day incubation, cell were trypsinized and seeded in 60 mm plates at a low density (500 cells/dish) and cultured for 14 days in a humidified 5% CO₂ incubator at 37°C. The cells were then rinsed with PBS and fixed and stained in a solution containing 0.25% crystal violet and 10% formalin (35% v/v) in 80% methanol for 15 minutes. Colonies were then counted and values are expressed as the fraction of cells surviving and normalized to the surviving fraction of control, which was set to a value of 100%.

Cells were lysed with NP40 buffer (150 mM NaCl, 1% NP-40, 50 mM Tris (pH:8.0)), boiled for 5 min and subjected to 6% polyacrylamide SDS gel electrophoresis. After electrophoresis, proteins were transferred to Immobilon-P or Immobilon-FL transfer membranes (Millipore). The membranes were then blocked with 5% nonfat dried milk in TBS-T (50 mM Tris-HCl, [pH8.0], 150 mM NaCl, 0.1% Tween 20) and then incubated with primary antibodies diluted in Universal Antibody Buffer (30% BSA, 0.2% NaH₃, 1% goat serum in TBS-T). The rabbit anti-FANCD2 antibodies (GeneTex, TX) were used in a 1:1000 dilution and the mouse anti-BRCA1 antibodies (Santa Cruz Biotechnology, CA) were used in a 1:100 dilution. After incubation with primary antibodies overnight at 4°C, membranes were washed with TBST and then incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies (Sigma, MO) at a 1:2000-fold dilution for 1 hr at room temperature. After rinses with TBS-T, immunoblotted proteins were captured on film by chemiluminescence.

Head and neck cancer cells were seeded on glass coverslips and incubated overnight before being treated with 2 mM PB for 3 days, 5 μM cisplatin for 24 hours or the combination of the two. Cells were then rinsed with PBS three times and fixed with 4% paraformaldehyde in PBS for 20 minutes at 4°C. The fixed cells were permeabilized with 2% Triton-X-100 in PBS for 8 minutes on ice. After blocking in Universal Antibody Buffer (30% BSA, 0.2% NaH₃, 1% Goat serum in TBS-T) for 30 minutes at room temperature, anti-FANCD2 and/or anti-BRCA1 antibodies were added at dilutions of 1:200 and 1:100, respectively. After 1 hr incubation at 37°C, cells were washed three times with PBSBT (PBS, 0.5% BSA, 0.05% Tween 20) and then incubated with rabbit AlexarFluor555 (red) and/or mouse AlexaFluor 488 (green) for 1 hr at 37°C. After incubation, cells were rinsed with PBSBT three times. Glass coverslips were mounted in Vectashield containing DAPI. Images were captured on a Nikon microscope and processed using Adobe Photoshop software. All the quantification of FANCD2 foci was performed in a blind fashion.