Targeting p53-mdm2 interaction using a potent mdm2 inhibitor to overcome cisplatin resistance in head and neck squamous cell carcinoma

3202 p53 is a transcription factor known to be implicated in more than half of all human cancers. p53 induces G1 cell cycle arrest and/or apoptosis. Murine double-minute protein (mdm2) is one of the most critical regulators of p53. mdm2 directly binds to p53 and blocks its transcriptional activity as well as acts as an E3 ubiquitin ligase promoting proteasomal degradation. As mdm2 inhibition could increase the magnitude of wild type p53 activation following DNA damage, the use of mdm2 antagonists could be a promising strategy for anti-cancer drug design. Resistance to chemotherapy is a problem encountered in the treatment of head and neck squamous cell carcinoma (HNSCC). We found previously that overexpression of Bcl-xL in combination with wild type p53 in HNSCC cell lines is associated with resistance to cisplatin. The purpose of this study was to look at the effectiveness of a recently developed non-peptide small molecule inhibitor of mdm2 along with cisplatin to see if these agents could be combined for more effective treatment of HNSCC tumors. To study this, we used two HNSCC cell lines: a parental, cisplatin sensitive cell line UM-SCC-74B (low Bcl-xL) and a stably transfected cisplatin resistant cell line UM-SCC-74BxL (very high Bcl-xL). Both cell lines have wild-type p53. Cell lines were treated with cisplatin alone, mdm2 inhibitor alone (MI-63), inactive control alone (MI-61), or in combination (sequentially). For combination treatments, cells were treated with cisplatin first for 4 hours, washed and 24 hours later treated with MI-63 or MI-61. The inhibition of cell growth following treatment was assessed by MTT assay. In the cisplatin sensitive UM-SCC-74B cell line, 49%, 50%, 26% viable cells as compared to control were seen in the cisplatin alone, mdm2 inhibitor alone or the combination treatment arm respectively. Similarly and more importantly, in the cisplatin resistant cell line UM-SCC-74BxL, the combination of cisplatin and mdm2 inhibitor was most effective in inhibiting cell growth (27% viable cells) as compared to treatment with cisplatin alone (70%) or mdm-2 inhibitor alone (50%). The inactive control, MI-61, had no effect on the growth of cells as compared to untreated cells. Similar results were obtained using the clonogenic survival assay. Significantly fewer clones were observed in the combination group in both cell lines as compared to the individual drug treated groups. Cell cycle analysis by propidium iodine staining showed that treating with mdm2 inhibitor alone caused cells to undergo G1 arrest. Cells seemed to arrested at G2/M in the combination treatment group, and this G2/M arrest was higher than the G2/M arrest observed in the cisplatin treated group alone. These results indicate that this mdm2 inhibitor in combination with cisplatin might have a great therapeutic potential in the treatment of chemotherapy resistant tumors with wild type p53.