Increased growth-inhibitory and cytotoxic activity of arsenic trioxide in head and neck carcinoma cells with functional p53 deficiency and resistance to EGFR blockade

Abstract

Background and purpose: Mutations in the p53 gene are frequently observed in squamous cell carcinoma of the head and neck region (SCCHN) and have been associated with drug resistance. The potential of arsenic trioxide (ATO) for treatment of p53-deficient tumor cells and those with acquired resistance to cisplatin and cetuximab was determined.

Material and methods: In a panel of 10 SCCHN cell lines expressing either wildtype p53, mutated p53 or which lacked p53 by deletion the interference of p53 deficiency with the growth-inhibitory and radiosensitizing potential of ATO was determined. The causal relationship between p53 deficiency and ATO sensitivity was evaluated by reconstitution of wildtype p53 in p53-deficient SCCHN cells. Interference of ATO treatment with cell cycle, DNA repair and apoptosis and its efficacy in cells with acquired resistance to cisplatin and cetuximab was evaluated.

Results: Functional rather than structural defects in the p53 gene predisposed tumor cells to increased sensitivity to ATO. Reconstitution of wt p53 in p53-deficient SCCHN cells rendered them less sensitive to ATO treatment. Combination of ATO with irradiation inhibited clonogenic growth in an additive manner. The inhibitory effect of ATO in p53-deficient tumor cells was mainly associated with DNA damage, G2/M arrest, upregulation of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) receptors and apoptosis. Increased activity of ATO was observed in cetuximab-resistant SCCHN cells whereas cisplatin resistance was associated with cross-resistance to ATO.

Conclusions: Addition of ATO to treatment regimens for p53-deficient SCCHN and tumor recurrence after cetuximab-containing regimens might represent an attractive strategy in SCCHN.

Figure 1. The p53 status interferes with the growth-inhibitory activity of ATO.

(A) Cells were left untreated or were irradiated with a single dose of 6 Gy. Four hours after IR, cells were harvested and their expression levels of p21 as a functional read-out for p53 transcriptional activity were determined by qRT-PCR. Relative quantification of p21 expression was done by normalization to the expression levels of porphobilinogen deaminase (PBGD) and to the untreated control using the ΔΔC_t-method. IR-induced p21 expression (mean fold induction and standard error) is presented. The p53-deficient and p53-proficient cell lines were grouped using >1.5-fold induction of p21 by IR as threshold. (B, C) Cells were seeded at a density of 300 cells/well in 12-well plates and incubated for a period of 10–14 days in the absence or presence of the indicated doses of ATO. Survival fractions for given treatments were calculated on the basis of the survival of non-treated cells. Each sample was done in triplicate. The results from at least three independent experiments with p53-deficient (B) and p53-proficient cell lines (C) are presented. The symbols for each individual cell line are given in the graphs.

Figure 2. Reconstitution of p53-deficient SCC9 cells with wt p53 renders them less sensitive to ATO treatment.

(A) SCC9-wtp53 cells were treated with increasing doses of Dox for 24 hs or (B) with a dose of 20 ng/ml Dox for the indicated time periods. The expression of p53 was detected by immunoblotting. Detection of HSC70 served as protein loading control. (C) Induction of p53 by treatment of SCC9-wtp53 cells with 20 ng/ml Dox was followed by upregulation of p21. (D) Reconstitution of wt p53 after Dox treatment inhibited the clonogenic growth of SCC9 cells (left graph). After correction for this growth-inhibitory effect of wt p53 itself, a significantly reduced sensitivity of SCC9-wtp53 cells to ATO treatment (75 nM) was observed (right graph). * p<0.05 (paired t-test).

Figure 3. ATO combined with IR inhibits the clonogenic survival of SCCHN cells in an additive manner.

Cells were seeded at a density of 300/well in 12-well plates. Twenty four hours after seeding, cells were left untreated or were treated with the indicated doses of ATO, IR or the combination of both. Non-irradiated cultures were processed along with irradiated cultures. Cells were incubated for up to 14 days. Survival fractions for given treatments were calculated on the basis of survival of non-treated cells and corrected for the effect of ATO alone. Each sample was done in triplicate. Mean values from at least three independent experiments are presented. The symbols for the different treatments are given in the top of the figure. The p53 genotype of each individual cell line is depicted in the graphs.

Figure 4. The growth-inhibitory effect of ATO in p53-deficient FaDu cells depends on dose and time and is associated with a cell cycle arrest in G2/M.

(A) FaDu cells were left untreated or were treated with increasing doses of ATO. After the indicated time, cells were harvested by trypsinization and cell numbers were counted. The mean cell numbers ± SEM of three independent experiments are presented. (B) Representative flow cytometry histograms of the cell cycle distribution are presented which were observed in p53-deficient FaDu (upper panel) and p53-proficient UD-SCC-2 cells (lower panel), either untreated (left) or after treatment with 500 nM ATO (right). (C) After treatment of FaDu cells and UD-SCC-2 cells with ATO, irradiation or the combination of both, cells were harvested and the relative numbers of cells in G2/M were determined by flow cytometry. The mean percentages ± SEM are presented. Asterisks mark samples for which significant differences compared to the untreated control (p<0.05) were observed.

Figure 5. ATO induces apoptosis in p53-deficient FaDu but not p53-proficient UD-SCC-2 cells.

(A) Representative flow cytometry histograms of sub-G1 analysis after treatment of p53-deficient FaDu (upper panel) and p53-proficient UD-SCC-2 cells with the indicated doses of ATO are presented. (B) FaDu and UD-SCC-2 cells were left untreated or were treated with ATO for 96 hs at the indicated doses. The mean percentages of cells with features of apoptosis, detected by annexinV-FITC/PI staining and flow cytometry analysis are presented. *significant differences compared to untreated control (p<0.05). (C, D) FaDu and UD-SCC-2 cells were left untreated or were treated with 5 µM of ATO for 48 hs. The relative changes in the mean percentages of cells expressing TRAILR1 and TRAILR2 (C) or displaying residual DNA double strand breaks, as determined by gamma-H2AX staining (D) are presented.

Figure 6. CDDP-resistant SCCHN cells show cross-resistance to ATO whereas cetuximab-resistant cells display increased ATO sensitivity.

The sensitivity of (A) cetuximab-resistant UT-SCC-9 cells (UT-SCC-9_CET-R) to ATO (left panel) or cetuximab (right panel) compared to their parental sensitive counterparts (UT-SCC-9_CET-S) was determined by the MTT assay. In addition, (B) the sensitivity of cisplatin-resistant FaDu cells (FaDu_CDDP-R) and parental FaDu_CDDP-S to ATO or CDDP treatment was determined. Briefly, cells were treated for 10 days with the drugs at the indicated concentrations. Cell viability was assessed by measuring the absorbance of the formazan solution. Each sample was analyzed in six technical replicates and the experimental series were repeated for four times. Survival fractions were calculated on the basis of untreated cells. The mean surviving fractions ± SEM are presented.